Protein therapy for corneal inflammation, epithelial wound healing, and photoreceptor degeneration

ABSTRACT

The present invention encompasses methods, compositions, and devices for treating an ocular disease, disorder or condition in a mammal. The invention includes polypeptides that possess anti-inflammatory, anti-apoptotic, immune modulatory and anti-tumorigenic properties, and their application in the treatment of eye disease, particularly diseases of the retina. In particular aspects, the invention includes administration of a therapeutic polypeptide such as a stanniocalcin family member protein for the treatment of an eye disease. Also included are fusion proteins and cells stimulated or modified to express the therapeutic polypeptides as set forth herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Provisional PatentApplication Ser. No. 61/508,587, filed Jul. 15, 2011, and is acontinuation-in-part application of PCT/US2011/000771, filed May 3,2011, which in turn claims priority to provisional application Ser. No.61/464,172, filed Feb. 28, 2011, and to provisional application Ser. No.61/330,735, filed May 3, 2010, the contents of each of which of theforegoing applications are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made, in part, using funds obtained from the U.S.Government (National Institutes of Health Grant No. R21EY020962), andthe U.S. Government therefore may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Eye disease is a significant cause of morbidity in the U.S. andthroughout the world. While therapies have improved over time for manyeye diseases, there remain many others for which therapy is of limitedor no benefit.

Diseases of the retina, including age-related macular degeneration(AMD), retinitis pigmentosa (RP), and diabetic retinopathy (DR), aremajor causes of legal blindness in the United States. AMD and RP shareclinical and pathologic features including end-stage blindness due tophotoreceptor and/or retinal pigment epithelium (RPE) cell death. DR isone of the most common complications of diabetes and the leading causeof blindness in people of working age in the United States and otherindustrialized countries. The estimated prevalence of diabeticretinopathy is nearly 30% and vision-threatening DR is nearly 5% in theadult population with diabetes (Zhang et al., 2010, JAMA 304; 649-656).The Eye Diseases Prevalence Research Group estimated in 2004 thatapproximately 4.1 million adults 40 years and older have diabeticretinopathy and that 1 of every 12 persons with diabetes in this agegroup has advanced, vision-threatening retinopathy. (The Eye DiseasesPrevalence Research Group, 2004, Arch Ophthalmol 122:552-563).

Despite adequate glycemic and blood pressure control and lipid-loweringtherapy, the number of DR patients continues to grow and therapeuticapproaches remain limited. There is a great need for the development ofnew strategies to prevent and treat DR. Studies have shown that DR hasprominent features of chronic, subclinical inflammation. Retinal vesselocclusion and degeneration is a typical feature of DR and is also acause of neovascularization. Mechanisms leading to capillarydegeneration may involve inflammatory cytokine-induced endothelial celldeath since inflammatory cytokines such as TNF-α and IL-1β are alsoknown to increase caspase 3 activity and potently induce endothelialcell apoptosis (Aveleira et al., 2010, Diabetes 59:2872-2882; DelMaschio et al., 1996, J Cell Biol 135:497-510). The apoptotic effect ofinflammatory cytokines may even be exaggerated in the presence ofhyperglycemia (Del Maschio, 1996).

Apoptosis of photoreceptors is a prominent feature in many retinaldegenerations, including AMD and RP. Reactive oxygen species (ROS) havebeen implicated in the initiation and/or exacerbation of cell death inAMD (Fletcher, et al., Ophthalmic Res., Vol. 44, No. 3, pgs. 191-198(2010); Beatty et al., Surv. Ophthalmol., Vol. 45, No. 2, pgs. 115-134(2000); Winkler, Mol. Vis., Vol. 5, pg. 32 (1999); Johnson, Curr. Opin.Cln. Nutr. Metab. Care, Vol. 13, pgs. 28-33 (2010); Totan et al., Curr.Eye Res., Vol. 34, No. 12, pgs. 1089-1093 (2009)) and antioxidantvitamin therapy is currently one of the mainstays of treatment innon-exudative AMD and RP (Johnson, 2010; Hartong et al., Lancet, Vol.368, pgs. 1795-1809 (2006)). Oxidative stress happens when ROS areoverproduced or when endogenous antioxidant systems are impaired.Mitochondria have long been recognized as a key source of ROS formationduring diabetes (Aiello et al., 1998, Diabetes Care 21:143-156).Mitochondria can generate ROS by leak of electrons to molecular oxygenat electron transport chain (ETC) complexes I, II and III (Jezek et al.,2005, Int J Biochem Cell Biol 37:2478-2503). In diabetes, the metabolismof glucose-derived pyruvate through the ETC complexes is increasedbecause of high-glucose concentration within cells, resulting insuperoxide overproduction by mitochondria (Giacco et al., 2010, Circ Res107:1058-1070). Although not curative, reduction of risk of disease andstabilization of vision have been observed following antioxidant vitamintherapy (Flectcher, 2010; Beatty, 2000; Johnson, 2010; Hartong, 2006).Moreover, two of the top modifiable risk factors in AMD—smoking andlight exposure—are thought to injure photoreceptors or RPE throughROS-mediated damage (Flectcher, 2010; Johnson, 2010).

Glaucoma is a group of diseases characterized by progressive optic nervedegeneration that results in visual field loss and irreversibleblindness. A critical element in the pathophysiology of all forms ofglaucoma is the death of retinal ganglion cells (RGCs). Strategies thatdelay or halt RGC loss have been recognized as potentially beneficial topreserve vision in glaucoma. In recent years, there has been anexponential increase in data regarding the molecular basis of RGC deathresulting from experimental models of acute and chronic optic nerveinjury as well as experimental glaucoma. A variety of molecular signalsand/or mechanisms which might act alone or in concert can promote RGCdeath. Possible molecular mechanisms include: neurotrophic factordeprivation, toxic pro-neurotrophins, activation of intrinsic andextrinsic apoptotic signals, mitochondrial dysfunction, excitotoxicdamage, and oxidative stress (Almasieh et al., 2012, Prog Retin Eye Res31:152-81).

Dry, atrophic (nonexudative) age-related macular degeneration, definedas progressive age-related degeneration of the macula associated withretinal pigment epithelial changes including atrophy and drusen, is acommon cause of vision loss in adults for which therapy is extremelylimited. Patients often develop a slow progressive loss of vision overtime. Vitamin therapies and other types of therapy are of limitedbenefit. More therapeutic options are available for patients withexudative age-related macular degeneration, which is associated withchoroidal or subretinal neovascularization. Nevertheless, despitetherapy such as laser or pharmacotherapy, many patients developprogressive vision loss. There is the need for therapies to reduce therisk of progressive vision loss in patients with both forms ofage-related macular degeneration.

In summary, there is the need for more effective treatment of manycommon diseases of the eye, such as diseases of the retina, cornea, andglaucoma.

SUMMARY OF THE INVENTION

The present invention is based in part upon the finding that certainpolypeptides possessing anti-inflammatory or anti-apoptotic propertiesare beneficial in the treatment of eye disease.

Certain embodiments of the present invention concern a method oftreating or preventing an eye disease in a subject that involvesadministering to a subject with an eye disease or at risk of developingan eye disease a pharmaceutically effective amount of a composition thatincludes an isolated polypeptide comprising a domain comprising astanniocalcin family member polypeptide, wherein the eye disease istreated or prevented. The subject can be, for example, a mammal, such asa rat, a mouse, a rabbit, a dog, a cat, a horse, a sheep, a goat, aprimate, or a human subject (such as a patient with an eye disease). Inparticular embodiments, the subject is a patient (human subject) withthe eye disease or at risk of developing the eye disease.

The stanniocalcin family member polypeptide may be, for example, astanniocalcin-1 (STC-1) polypeptide that has at least 95% sequenceidentity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,or SEQ ID NO:11. In some embodiments, the STC-1 polypeptide has between95% and 99% sequence identity with one of the aforementioned polypeptidesequences. In a particular embodiment, the stanniocalcin family memberpolypeptide has at least 95% sequence identity to SEQ ID NO:1; and in aspecific embodiment, the stanniocalcin family member polypeptideconsists of SEQ ID NO:1.

The disease may be any eye disease, but in particular aspects is adisease of the retina, a disease of the cornea, or glaucoma. The diseaseof the retina may be, for example, a retinal degeneration such asmacular degeneration or retinitis pigmentosa. The macular degenerationmay be age-related macular degeneration. The age-related maculardegeneration may be atrophic nonexudative age-related maculardegeneration. The disease of the retina may also be diabeticretinopathy. The diabetic retinopathy may be proliferative diabeticretinopathy (retinopathy associated with neovascularization of theretina) or nonproliferative diabetic retinopathy (diabetic retinopathywithout neovascularization but with other findings associated withdiabetic retinopathy such as dot and blot hemorrhages andmicroaneurysms). The diabetic retinopathy may also include diabeticmacular edema. The disease may be any disease associated with elevatedintraocular pressure. Nonlimiting examples include chronic open angleglaucoma, angle closure glaucoma, and pigmentary glaucoma.

The composition may be administered using any method known to those ofordinary skill in the art. Nonlimiting examples include topical,subconjunctival, sub-Tenon's, intravitreal, subretinal, or injectioninto the anterior chamber of the eye of a subject. Other modes ofadministration include systemic administration, including intravenousadministration as well as oral administration. In a specific embodiment,the composition is administered intravitreally.

In some aspects the stanniocalcin family member polypeptide is astanniocalcin-2 (STC-2) polypeptide that has at least 95% sequenceidentity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20. Thestanniocalcin family member polypeptide may have between 95% and 99%sequence identity to any of the aforementioned stanniocalcin-2 familymembers. In more particular aspects, the stanniocalcin family membercomprises SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

Certain other embodiments involve administering to a subject apharmaceutically effective amount of a composition that includes cellsto treat an eye disease, wherein the cells have been stimulated orgenetically modified to overexpress a stanniocalcin family memberpolypeptide compared to a cell of the same type that has not beenstimulated or genetically modified. While any cell type is contemplated,in particular embodiments the cell is a mesenchymal stem cell (MSC), aretinal pigment epithelial cell, a corneal or conjunctival epithelialcell, or a limbal stem cell. In particular aspects, the stanniocalcinfamily member polypeptide has at least 95% sequence identity to SEQ IDNO:1 or SEQ ID NO:12. The polypeptide may have between 95% and 99%sequence identity to SEQ ID NO:1 or SEQ ID NO:12. The mesenchymal stemcells may be autologous or allogeneic. Autologous cells may be harvestedfrom the subject using any method known to those of ordinary skill inthe art, such as by venipuncture or bone marrow aspiration. The cellsmay be genetically modified in situ to express a stanniocalcin familymember polypeptide as set forth herein, and then administered to thesubject using any method known to those of ordinary skill in the art. Inparticular embodiments for the treatment of retinal disease such asretinal degeneration or age-related macular degeneration, the cells areadministered by intravitreal or subretinal injection or via an oculardrug delivery device designed for insertion into the vitreous cavity.

Other aspects of the invention pertain to a method of treating orpreventing an eye disease in a subject, that involves administering to asubject with an eye disease or at risk of developing an eye disease apharmaceutically effective amount of a composition that comprises anisolated polypeptide comprising a domain comprising a Tumor NecrosisFactor-Inducible Gene 6 (TSG-6) polypeptide, wherein the eye disease istreated or prevented. Non-limiting examples of such polypeptides includethose having at least 95% sequence identity to SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25. The polypeptide mayhave between 95% and 99% sequence identity to SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25. In particularaspects, the eye disease is a disease of the cornea. In certain aspects,the subject has had an injury or condition resulting in a defect of thecorneal epithelium, or has had corneal transplantation or other cornealsurgery.

Other aspects of the invention involve administering to the subject apharmaceutically effective amount of a composition that includes cells,wherein the cells have been stimulated or genetically modified tooverexpress a TSG-6 family member polypeptide. The cells can be any ofthe foregoing cell types for delivery of a stanniocalcin family memberpolypeptide.

The present invention also includes methods of treating a subject withretinal degeneration comprising administering to a subject with retinaldegeneration a pharmaceutically effective amount of a compositioncomprising mesenchymal stem cells, wherein the injection is intravitrealor subretinal. In particular embodiments, the retinal degeneration isretinitis pigmentosa or age-related macular degeneration. In someembodiments, the stem cells have been genetically modified tooverexpress a stanniocalcin family member polypeptide as set forthherein. In a particular embodiment, the stanniocalcin family memberpolypeptide is an STC-1 polypeptide. Overexpression of the stanniocalcinfamily member polypeptide is overexpression compared to the expressionof the same stanniocalcin family member polypeptide by mesenchymal stemcells that have not been genetically modified.

Also included as part of the invention are kits that include (a) apharmaceutically effective amount a stanniocalcin family memberpolypeptide or a TSG-6 family member polypeptide in one or more sealedvials. The stanniocalcin family member polypeptide and TSG-6 polypeptidemay include any of the sequences previously set forth. The polypeptidemay have between 95% and 99% sequence identity to any of the foregoingsequences. In a particular aspect, the kit includes a pharmaceuticallyeffective amount of a STC-1 polypeptide that has at least 95% sequenceidentity to SEQ ID NO:1. In further aspects, the kit includes a syringe.For example, the syringe may be a tuberculin syringe. The kit mayinclude a 25-gauge needle or a 30-gauge needle. The kit may optionallyinclude instructions for use of the polypeptide either written on apaper or in a computer-readable format. The kit may optionally furtherinclude a 1 cc syringe or a 2 cc syringe. In a particular aspects, thestanniocalcin family member polypeptide comprises SEQ ID NO:1. In aparticular aspect, the polypeptide is in a lyophilized form, and the kitfurther includes instructions for reconstituting the lyophilizedpolypeptide into a carrier for administration to a subject. For example,the carrier may be sterile water, normal saline, or phosphate bufferedsaline. The carrier may be comprised in one or more separate vials. Insome aspects, the kit includes a polypeptide as set forth hereincomprised in an ophthalmic drug delivery device, including abiodegradable drug-eluting device.

Other embodiments of the present invention concern methods of treatingan eye disease comprising administering to a subject a pharmaceuticallyeffective amount of a polynucleotide expressing a STC-1, STC-2, or TSG-6polypeptide. The STC-1, STC-2, or TSG-6 polypeptide may be selected fromany of the aforementioned polypeptide sequences. In some aspects, thepolynucleotide is comprised in an expression cassette wherein thepolynucleotide is operatively coupled to a promoter that facilitatesexpression of the polypeptide in a target cell. In some aspects, theexpression cassette is comprised in a vector, such as a cell (such as aMSC), viral vector, a liposome, or a nanoparticle.

Fusion proteins and polynucleotides encoding them (RNA, and DNA, such asDNA polynucleotides in expression vectors) are also contemplated as partof the invention. A fusion protein is a single polypeptide sequencecreated through the joining of two or more genes which originally codedfor separate polypeptides, with functional properties derived from theoriginal polypeptides. The fusion proteins include a first domain thatincludes a stanniocalcin family member polypeptide or a TSG-6 familymember polypeptide and a second domain comprising a second therapeuticpolypeptide or a carrier polypeptide to facilitate transfer of thefusion protein into a cell. In a particular aspect, the secondtherapeutic polypeptide is a CD59 polypeptide or an antiangiogenicpolypeptide. The sequence of human CD59 is provided in SEQ ID NO:26 andis associated with GenBank accession number CAG46523. Nonlimitingexamples of angiostatic polypeptides include ranibizumab, endostatin,bevacizumab, or aflibercept. Additional non-limiting examples of agentswhich may be present in the fusion protein or in other covalent ornon-covalently associated complexes with a stanniocalcin family memberpolypeptide or a TSG-6 family member polypeptide are Lucentis, Macugen,Pegaptanib, Ranibizumab, Eylea, Verteporfin, Visudyne, an angiostaticcortisene formulation, such as anecortave acetate suspension (RETAANE).Nonlimiting examples of carrier polypeptides include poly-Arg, aTat-derived amino acid sequence and Drosophila Antennapediahomeodomains. Numerous examples of carrier polypeptides are set for inU.S. Pat. No. 7,939,493, which is herein specifically incorporated byreference in its entirety. In this patent they are designated as“carrier peptides.” A “peptide” for purposes of the present patentapplication is an example of a “polypeptide” and the terms are usedinterchangeably herein. Other embodiments include a polynucleotideencoding a fusion protein of the present invention.

Ophthalmic drug delivery devices for intravitreal or subconjunctivaldelivery of the stanniocalcin family member polypeptide, a TSG-6polypeptide, a fusion protein, or a stimulated or genetically modifiedcell as set forth herein are also contemplated as part of the presentinvention. The ophthalmic drug delivery device may comprise an effectiveamount of any of the foregoing polypeptides, fusion proteins, and cellsset forth herein. In some embodiments, the polypeptide has between 95%and 99% sequence identity to any of the foregoing sequences. Thepolypeptide may be enclosed in a reservoir or in contact with a surfaceof the drug delivery device. In some aspects, the drug delivery deviceis a scleral-fixated nonbiodegradable implant. In other embodiments, thedrug delivery device is a biodegradable implant designed to befree-floating in the vitreous cavity. In some embodiments, the drugdelivery device further comprises cells that express the STC-1polypeptide in the reservoir or in contact with a surface of the drugdelivery device. In particular aspects, the ophthalmic drug deliverydevice is OZURDEX™, VITRASERT™, I-VATION™, TRETISERT™, or ILUVIEN™.Other non-limiting examples of drug delivery devices include OCUSERT®,collagen shields, or a delivery device comprising polyacrylic acid,polyvinyl alcohol, silicone elastomer, hydroxy propyl cellulose, ethylcellulose, cellulose acetate phthalate and polymethacrylic acid, orhyaluronic acid.

Also included are pharmaceutical compositions for ophthalmic deliverycomprising a therapeutically effective amount of at least onetherapeutic polypeptide selected from any of Tables 1, 2, or 3, or apolypeptide that has at least 95% sequence identity to a protein setforth in any of Tables 1, 2, or 3 or a polypeptide that has between 95%and 99% sequence identity to any of the polypeptides set forth in Tables1, 2, or 3. The pharmaceutical composition may optionally comprise oneor more additional therapeutic agents suitable for ophthalmic delivery.Non-limiting examples of additional therapeutic agents includeantibiotics, anti-inflammatory agents, anti-proliferative agents,anti-neovascular agents (such as agents which antagonize the function ofneovascular growth factors (i.e., vascular endothelial growth factor(VEGF), endothelial cell surface receptors, and/or extracellular matrix(ECM) proteins, which are important mediators of neovascularization),anti-viral agents (i.e., idoxuridine, vidarabine, trifluorothymidine),beta-andrenergic blockers (timolol maleate, levobunolol),corticosteroids, retinoic acid formulations, vitamins, topicalanesthetics (i.e., proparacaine hydrochloride, tetracainehydrochloride), and the like. Other examples include bevacizumab(Avastin), pegaptanib (Macugen), Lucentis (ranibizumab), verteporfin(Visudyne), and CD59.

Additional non-limiting, particular examples of therapeutic agents thatcan be included in the pharmaceutical compositions of the inventioninclude Avastin (bevacizumab), Lucentis (ranibizumab), cyclosporine,erythromycin, tobramycin, gentamcyin, fluoroquinolones,medroxyprogesterone acetate, hypromellose, carboxymethylcellulosesodium, and olopatadine. The compositions may be comprised in a vial,such as in a kit. The therapeutic agent may be fused or linked to thetherapeutic polypeptides described herein.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiment(s) which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown.

FIG. 1, comprising FIGS. 1A through 1C, is a series of images depictinginduction of corneal inflammation and neovascularization, one weekpost-injury. FIG. 1A is an image demonstrating that on slit lampexamination, corneal inflammation with active new vessels was observed.FIG. 1B is an image of immunofluorescent staining for VEGF showing themarked increase in VEGF expression in cornea. VEGF as green and thenuclei counterstained as blue. FIG. 1C is an image depictingHematoxylin-eosin staining revealing that the cornea was denselyinfiltrated with inflammatory cells.

FIG. 2, comprising FIGS. 2A and 2B, is a series of images depictingapplication of cells to cornea. FIG. 2A is an image of a 6-mm-diameterhollow plastic tube placed to keep the eye open and the cells or mediaapplied to the cornea into a customized applicator. FIG. 2B is an imagedepicting engraftment of MSCs in the cornea confirming by identificationof PKH26-labeled cells in corneas by fluorescein microscopy.

FIG. 3, comprising FIGS. 3A through 3L, is a series of images depictingphotography of cornea one (FIGS. 3A-3D), two (FIGS. 3E-3H), and threeweeks (FIGS. 3I-3L) post-injury. With time, neovascularization andopacity markedly decreased in the corneas with MSCs (FIGS. 3D, 3H, 3L)or MSC-CM media three times (FIGS. 3C, 3G, 3K), while increased in thecontrol (FIGS. 3A, 3E, 3I). Corneas treated with MSC-CM once (FIGS. 3B,3F, 3J) showed the intermediate outcome.

FIG. 4, comprising FIGS. 4A through 4D, is a series of images depictinghematoxylin-eosin staining of cornea three weeks post-injury. Control(FIG. 4A) and corneas treated with MSC-conditioned media once (FIG. 4B)were densely infiltrated with inflammatory cells in the stroma andgoblet cells in the epithelium. The infiltration was markedly reduced inthe corneas with MSC-conditioned media three times (FIG. 4C) or MSCs(FIG. 4D).

FIG. 5, comprising FIGS. 5A through 5D, is a series of imagesdemonstrating inflammation-related cytokine expression evaluated byELISA. IL-2 and IFN-γ were repressed in the corneas treated with MSCs orMSCs-conditioned media three times (MSC-CM II) compared to the control.

FIG. 6, comprising FIGS. 6A through 6D, is a series of images depictingreal-time PCR for angiogenesis-related cytokines. Upregulation of TSP-1was observed in the corneas treated with MSC or MSC-conditioned mediathree times (MSCCM II), compared to the control. MMP-2 and MMP-9 weredownregulated in the MSC group. There were no differences in theexpression of VEGF. Values were expressed as folds relative to freshcorneas without an injury.

FIG. 7 is an image depicting cytotoxicity test of human cornealepithelial cells (HLECs) after chemical damage. When cultured withhMSCs-derived medium for 48 hours, damaged HLECs were significantlydecreased compared to HLECs without hMSCs-conditioned medium.

FIG. 8 is an image depicting cytokine secretion evaluated by ELISA. Theexpression of VEGF, MMP-9, MMP-2, and TSP-I were quantified in variouscocultures of hMSCs/hCECs/hPBMCs. The hCECs were prepared aftertreatment with 15% ethanol for 30 sec. Data represent at least threeexperiments.

FIG. 9, comprising FIGS. 9A through 9F, is a series of images depictingassays for the fate of hMSCs infused into mice. FIG. 9A is an imagedepicting clearance of human Alu sequences from blood after IV infusionof about 2×10⁶ hMSCs into mice. Values are mean+/−_(−/−)SD; n=6. FIG. 9Bis an image depicting standard curves for real time PCR assays of humanAlu sequences in 7 organs. Values indicate ΔΔCt for primers formouse/human GAPDH genes and Alu sequences on same samples. FIG. 9C is animage depicting tissue distribution of human Alu sequences 15 mm afterIV infusion of about 2×10⁶ hMSCs into mice. Values are mean+/−SD; n−6.FIG. 9D is an image depicting standard curves for real time RT-PCRassays of human mRNA for GAPDH. Values indicate ΔΔCt for primers formouse/human GAPDH genes and cDNA for human-specific GAPDH on samesamples. FIG. 9E is an image depicting kinetics of hMSCs in lung and 6other tissues after IV infusion of about 2×10⁶ hMSCs, Values aremean+/−SD; n−6. FIG. 9F is an image depicting appearance of hMSCs inheart after IV infusion of about 1×10⁶ hMSCs one day after permanentligation of the left anterior descending coronary artery.

FIG. 10, comprising FIGS. 10A through 10F, is a series of imagesdepicting activation of hMSCs to Express TSG-6. FIG. 10A is an imagedepicting realtime RT-PCR for human-specific mRNA in lung 10 hr after IVinfusion of 2×10⁶ hMSCs. Values are fold increase over values forcultured hMSCs, normalized by ΔΔCt for hGAPDH. Symbols: hMSCs con,sample of hMSCs added to lung from control mouse before extraction ofRNA; hMSCs IV 1 and 2, samples from lungs of 2 mice 10 hr after IVinfusion of hMSCs. FIG. 10B is an image depicting real-time RT-PCR forhuman TSG-6 in mouse lung. About 2×10⁶ hMSCs were infused IV into naĩvemice (IV-nor) or mice at 1 hr after Ml (IV-MI) and lungs were recovered0.25 hr to 24 hr later. Values are +/−SD; n−2 or 3 for normal mice; n=6for MI mice. FIG. 10C is an image depicting real-time RT-PCR for TSG-6in hMSCs and human fibroblasts from the same donor incubated inserum-free medium with 10 ng/ml TNF-α for 24 or 48 hr. Results with twopassages of the same cells are shown. Values are +/−SD; n−3. FIG. 10D isan image depicting ELISAs for TSG-6 in medium from hMSCs and humanfibroblasts incubated in serum-free medium with 10 ng/ml TNF-a for 48hr. Values are +/−SD; n=3. FIG. 10E is an image depicting real-timeRT-PCR assays for TSG-6 of control hMSCs (Con), hMSCs treated withtransfection reagents only (no siRNA), hMSCs transfected with ascrambled siRNA (scr siRNA) or hMSCs transduced with TSG-6 siRNA (TSG-6siRNA). Cells were incubated with or without 10 ng/ml TNF-a for 6 hr.Values are +/−SD; n=3. FIG. 10F is an image depicting ELISAs for TSG-6in medium after incubation of cells with or without TNF-α for 48 hr.Symbols: as in FIG. 10E. Values are +/−SD; n=3.

FIG. 11, comprising FIGS. 11A through 11E, is a series of imagesdepicting assays of serum and heart. FIG. 11A is an image depicting anassay for cardiac troponin I in serum 48 hr after MI. Values are +/−SD;**p<0.01 with n=3 (Normal) or 6 mice (MI) per group. FIG. 11B, is animage depicting plasmin activity in serum 48 hr after MI. Symbols:Normal, naĩve mice; MI only; hMSCs, 2×10⁶ hMSCs infused IV I hr afterMI; scr siRNA, 2×10⁶ hMSCs transduced with scrambled siRNA infused IV 1hr after MI; TSG-6 siRNA, 2×10⁶ hMSCs transduced with TSG-6 siRNAinfused IV 1 hr after MI; rhTSG-6, 30 μg rhTSG-6 protein infused IV 1 hrand again 24 hr after MI. Values are +/−SD; p<0.01 with n=3 mice pergroup. N.S.=not significant. FIG. 11C, is an image depicting heartsassayed for pro- and active-matrix MMP9 on a gelatin zymogen gel 48 hrafter MI. Image is reversed. Symbols: as in FIG. 11B. FIGS. 11D and 11Eare images depicting granulocyte and monocyte infiltration in the heart48 hr after MI. Sections stained with anti-Ly-6G and Ly-6C. Symbols: asin FIG. 11B except 100 μg rhTSG-6 protein was infused IV 1 hr and again24 hr after MI. Magnification×4. Scale bars, 250 μm. Values are +/−SD;n=3 or 4 for each group. **p<0.001; N.S.=not significant.

FIG. 12, comprising FIGS. 12A through 12F, is a series of imagesdepicting assays of Infarct Size 3 wk after MI. Each heart was cut fromthe apex through the base into over 400 sequential 5 μm sections andstained with Masson Trichrome. Every 20th section is shown from typicalspecimens. FIG. 12A is an image depicting MI. Heart with no treatment.FIG. 12B is an image depicting MI+hMSCs. 2×10⁶ hMSCs infused IV 1 hrafter MI. FIG. 12C is an image depicting MI+scr siRNA, 2×10⁶ hMSCstransduced with scrambled siRNA infused IV 1 hr after MI. FIG. 12D is animage depicting MI+TSG-6 siRNA. 2×10⁶ hMSCs transduced with TSG-6 siRNAinfused IV 1 hr after MI. FIG. 12E is an image depicting MI+hTSG-6 100μg rhTSG-6 protein infused IV 1 hr and again 24 hr after MI. FIG. 12F isan image depicting Infarct size measurements (%) obtained by midlinelength measurement from every 10^(th) section of the infarct area for atotal of 20 sections per heart (Takagawa et al., 2007). Values are+/−SD; n−3 or 4 mice per group; ***p<0.0001 compared to MI controls;N.S. not significant compared to MI controls; *p<0.05 for MI+MSCs versusMI+rhTSG-6.

FIG. 13 is an image demonstrating that STC-1 was required and sufficientfor reduction of apoptosis of lung epithelial cell line made apoptoticby incubation at low pH in hypoxia. Upper left and right: Cultures ofA549 cells became apoptotic when incubated for 24 hours in 1% oxygen atpH 5.8 or 5.5. However, coculture of A549 cells in transwells with MSCsreduced the apoptosis. Lower left: Apoptosis of A549 cells was inhibitedby rhSTC-1, and the effects were reversed by anti-STC-1 antibodies.Lower right: MSCs transduced with siRNA for STC-1 were less effectivethan control MSCs in decreasing apoptosis of A549 cells in the transwellexperiment.

FIG. 14 is a schematic of a strategy to search for additional, noveltherapeutic factors produced by hMSCs in response to corneal injury.

FIG. 15 is a series of images demonstrating that conditioned medium frompre-activated MSCs and rhSTC-1 had the greatest effects in hMSCsimproving the viability, increasing the proliferation, and inhibitingthe apoptosis of damaged hCEPs.

FIG. 16, comprising panels A through panel F, is a series of imagesdemonstrating that intracameral injection of TSG-6 (2 ug) decreasedcorneal opacity and neovascularization in cornea after injury. FIG. 16,panel A-panel F are photographic images of the cornea. FIGS. 16A-16C areimages depicting PBS-treated control.

FIG. 16 panel D-16 panel F are images depicting TSG-6-treated cornea.

FIG. 16 panel A, 16 panel D depict postoperative day 3.

FIG. 16 panel B, 16 panel E depict postoperative day 7.

FIG. 16 panel C, 16 panel F depict postoperative day 21. Bottom frames:Clinical evaluations of opacity (left frame) and neovascularization(right frame) of the cornea.

FIG. 17, comprising Figures panel A through panel D, is a series ofimages demonstrating intracameral injection of TSG-6 (2 ug) decreasedthe infiltration of neutrophils and production of MMP-9 in cornea afterinjury. (FIGS. 17 panel A-17 panel D) Hematoxylin-eosin staining ofcornea. (FIGS. 17 panel A, 17 panel B) PBS-treated cornea. (FIGS. 17panel C, 17 panel D) TSG-6-treated cornea. (FIGS. 17 panel A, 17 panelC) Postoperative day 3. (FIGS. 17 panel B, 17 panel D) Postoperative day21. (FIG. 17 upper graph) Myeloperoxidase assay. (FIG. 17 lower rightgraph) Gel zymography for MMP-9. (FIG. 17, Lowe left graph) ELISA fortotal and active MMP-9.

FIG. 18 is a graph depicting correlation between clinical opacity andMPO amount in cornea at post-injury 3 days.

FIG. 19, comprising FIGS. 19A through 19D, is a series of imagesdemonstrating that TSG-6 up to the concentration of 2 ug is effective inreducing corneal opacity, inflammation, and MMP-9 production. (FIGS.19A-19D) Photography of cornea. (FIG. 19A) PBS-treated cornea. (FIG.19B) TSG-6 0.02 ug-treated cornea. (FIG. 19C) TSG-6 0.2 ug-treatedcornea. (FIG. 19D) TSG-6 2 ug-treated cornea. (FIG. 19E) Myeloperoxidaseassay and clinical grading of opacity. (FIG. 19F) Gel zymography andELISA for MMP-9.

FIG. 20 is an image depicting real time PCR for inflammatory cytokinesand chemokines. RQ: relative gene expression. Comparisons are betweenvehicle treatment (PBS) and TSG-6 treatment (2 micrograms) onpost-operative days (POD) 3, 7, and 21.

FIG. 21 is an image depicting ELISA for cytokines and chemokines.Conditions as in FIG. 20.

FIG. 22 is an image demonstrating that TSG-6 delayed the timeneutrophils started to infiltrate and arrived at its peak as well asdecreased the amount of infiltrated neutrophils. The expression patternof chemokines and cytokines showed similar kinetics. Lower two frames:Blood levels of MPO and leukocytes (WBC).

FIG. 23. Two injections of STC-1 rescued retinal degeneration in therhodopsin mutant transgenic rat. Upper frame: representative posteriorsegment histology showed thickened ONL in STC-1 treated eyes compared toUI controls. The ONL is the outer nuclear layer of the retina thatcontains the nuclei of the rods and cones. Lower frame: representativeplot of ONL layer thickness taken from a total of 54 measurements (27superior retina and 27 inferior retina) demonstrated STC-1 significantlyimproved ONL thickness compared to UI controls.

FIG. 24. Age-related loss of mRNAs for photoreceptors in RCS rat.qRT-PCR analysis for the photoreceptor genes: rhodopsin, phosducin,neural retina leucine zipper, and recoverin. Expression of these genesdecreases over time in the RCS rat. For qPCR methods see Lee, 2008.

FIG. 25. Rescue of mRNAs for photoreceptors by intravitreal injection ofSTC-1 in RCS rat. qRT-PCR analysis for photoreceptor genes was conductedas described with respect to FIG. 24.

FIG. 26 MSCs survive in the vitreous cavity following injection. LeftPanel: standard curve with human specific qRT-PCR for human GAPDH mRNAas a reflection of viable MSCs (see Lee, 2009 for Methods). Varyingnumbers of human MSCs added to whole globe just before RNA wasextracted. Right Panel: Recovery of viable human cells 4 days afterintravitreal injection of 100,000 human MSCs.

FIG. 27. Activation of expression of STC-1 by culture of human MSCs inhanging drops so that the cells coalesced into spheroids. High densitymonolayer (Adh High), spheroids (Sph 25 k), and spheroid derived MSCs(Sph 25 k DC) were transferred to 6 well plates containing 1.5 mlcomplete culture medium (CCM) and either 200,000 MCSs from high densitycultures, eight 25 k spheroids, or 200,000 MSCs. After 24 hours, mediumwas recovered for ELISAs and cells were lysed for protein assays. Figureadapted from (Bartosh, 2010). The results demonstrated an over 20-foldincrease in secretion of STC-1 by spheroid MSCs (Sph 25 k-Adh, Sph 25k-Non adh, or Sph 25 k DC-Adh) compared to standard monolayer culturesof MSCs (Adh High-Adh).

FIG. 28. Anti-apoptotic effects of STC-1 in cultures of RPE cells.Treatment with STC-1 (250 ng/mL) one hour following injury of ARPE-19with 450 μM H₂O₂ reduced expression of a pro-apoptotic gene (caspase3/7), cell death (Annexin V & PI staining cells) and improved cellviability (increased activity of the mitochondrial enzyme MTT).Detection of caspase activity was performed as described previously inSharma, et al., Invest. Ophthalmol. Vis. Sci., Vol. 49, No. 11, pgs,5111-5117 (2008), annexin/PI quantification as described previously inBartosh, 2010, and MTT conversion was measured as described previouslyin Mester, et al., J. Mol. Neurosci., (2010).

FIG. 29. Anti-apoptotic effect of STC-1 with intravitreal injection inRCS rats. Gene expression of BAX, a transcript that encodes apro-apoptotic protein, was reduced significantly by STC-1 as quantifiedby qRT-PCR.

FIG. 30. Immediately after chemical and mechanical injury to mousecorneas, either PBS was administered to the mice intravenously orintraperitoneally, or TSG-6 (2 mg/5 ml) was applied to the surface ofthe mouse corneas. Lateral tarsorrhaphies then were performed on theeyes of the mice. Three days later, the corneas were extracted andmyeloperoxidase (MPO) ELISA assays were performed.

FIG. 31. Early events in the cornea after injury. A. The neutrophilinfiltration occurred in the two phases: 1) a small initial phase thatbegan within about 15 min, and reached a plateau level at 4 h (Phase I)and 2) a much larger infiltration of neutrophils with a peak at 24 to 48h (Phase II). B. Based on the temporal pattern of expression inmicroarrays, the up-regulated genes in the injured cornea were dividedinto three groups. C. Real time RT-PCR analysis of representative genesin each group. The group A genes preceded Group B and C genes in mRNAexpression. D. Microarray heat map of genes from the corneas 4 h and 24h after injury. Gene ontology categories and the number of genesup-regulated (red) or down-regulated (blue)>2-fold are indicated. Basedon the expression pattern, genes were categorized into three groups:genes whose expression increased rapidly early after injury andthereafter decreased (Group A), genes that were expressed at steadylevels (Group B), and genes increased gradually after injury (Group C).

FIG. 32. Expression patterns of secretoneurin (SN) and HSPB4 in theinjured cornea. A, E. Western blot of SN and HSPB4 in the cornea. SN wasreleased into the cornea immediately after injury, and HSPB4 reached apeak at 4 h. B, F, G. ELISA of SN and HSPB4 in the serum and cornea. SNwas released into the cornea and the serum within 0.25 h of the injury.HSPB4 was released into the extracellular space as measured in thesupernatants of the ex vivo culture of corneas after 2 to 4 h. C, HImmunohistochemistry of SN and HSPB4 in the cornea. D, I. Real timeRT-PCR of neuropeptides and crystallins. Among neuropeptides andcrystallins analyzed, SN and HSPB4 showed the highest expression in theinjured cornea. J. In response to necrotic extracts, keratocytes inculture expressed HSPB4. K. As measured by aconitase activity, oxidativestress was generated in the cornea by injury. L. The hydrogen peroxideincreased the expression of HSPB4 in keratocytes. M. Temporal expressionof IL-6, IL1β, CXCL1, and CCL2 in the cornea. ELISAs demonstrated thatthe expression of proteins of Group B (IL-6) and Group C (IL-1β, CXCL1,and CCL2) genes paralleled gene expression as assayed for mRNAs withmicroarrays and real time RT-PCR assays.

FIG. 33. SN reproduced the Phase I inflammatory response, and HSPB4reproduced both the Phase I and Phase II. A. The injection of therecombinant SN induced the early infiltration of neutrophils of Phase I,but not of Phase II. B, C. HSPB4 injection induced the Phase I and PhaseII responses accompanied by corneal opacity and neutrophil infiltrationas shown in hematoxylin-eosin staining and immunostaining for neutrophilelastase of the region of the cornea into which HSPB4 was injected. D.The topical application of the calcium channel blocker Diltiazeminhibited significantly the Phase I response in corneal injury tochemical injury. E. The subconjunctival injection of polyclonal (pAb) ormonoclonal (mAb) antibodies to HSPB4 decreased significantly theneutrophil infiltration in Phase II, compared to isotype control(IgG)-injected group. F. The amounts of SN and HSPB4 released into thecornea were dependent on the severity of injury as measured by real timePCR of SN and HSPB4 in the injured cornea and ELISA for SN and HSPB4 inthe serum or cornea. The concentrations of mRNAs and proteins werehigher in the cornea or serum by severe injury (30 sec ethanol andscraping), compared to mild injury (15 sec ethanol and scraping). G.After subconjunctival injections of clodronate-encapsulated liposome(Cl₂ MDP-LIP) on day 2 (i.e., 2 days before injury) and day 0(immediately after injury), sections of the rat cornea were stained withhematoxylin-eosin (H&E), or antibodies to CD11b and CD68 to identifymacrophages. The structure of the cornea on H&E was not affected by Cl₂MDP-LIP. CD11b- and CD68-positive cells in the cornea, however, weredecreased significantly by Cl₂MDP-LIP compared to PBS-encapsulatedliposome-injected controls (PBS-LIP).

FIG. 34. HSPB4 activated macrophages through TLR2/NK-kB signaling. A.HSPB4 did not induce the Phase II response when corneal macrophages weredepleted by subconjunctival injection of liposome-encapsulatedclondronate (Cl₂MDP-LIP). B. An intracameral injection of TSG-6, aninhibitor of TLR2/NF-kB signaling, suppressed the Phase II inflammatoryresponse of the cornea after injury. C. TSG-6 treatment also decreasedsignificantly the neutrophil infiltration in Phase II in the corneainjected with HSPB4. D. Macrophages were activated to expresspro-inflammatory cytokines when incubated with necrotic extracts of thecornea. Blocking HSPB4 with polyclonal (pAb) or monoclonal (mAb)antibodies negated significantly the effects of necrotic cornealextracts on macrophage activation. E. The addition of recombinant HSPB4activated macrophages in culture in a dose-dependent manner. F. HSPB4induced the translocation of NK-kB from the cytoplasm into nucleus inmacrophages. G. Necrotic extracts of the cornea also stimulate theTLR2/NK-kB pathway in the reporter cell expressing TLR2 (HEK-TLR2), butantibodies to HSPB4 partially inhibited the effects. H, I, J.Recombinant HSPB4 stimulated NF-kB signaling in the cell line expressingTLR2 or TLR4 (HEK-TLR4) in a dose-dependent manner, while it had noeffect in the cell without either receptor (HEK-null). K. Murinemacrophages (RAW 264.7) were incubated with Group A molecules (HSPB4,HSPB5, or β-crystallin) and evaluated for expression of pro-inflammatorycytokines by real-time RT-PCR. Neither HSPB5 nor 3-crystallin activatedmacrophages, while HSPB4 induced remarkably the expression ofpro-inflammatory cytokines in macrophages. In contrast, heat-treatedHSPB4 (boiling, 20 min) did not activate macrophages in culture,indicating that HSPB4, and not contaminating pyrogens, induced themacrophage activation. Human embryonic kidney cells expressing TLR-2(HEK-TLR2) were incubated with HSPB5 or β-crystallin and evaluated foractivation of NK-kB signaling. Neither HSPB5 nor β-crystallin activatedTLR2/NK-KB signaling. L. Sterile injury was made to the rat cornea afterresident macrophages were depleted by subconjunctival injections ofclodronate-encapsulated liposome (Cl₂ MDP-LIP) on day 2 (2 days beforeinjury) and day 0 (immediately after injury). The cornea was evaluatedfor neutrophil infiltration by assays for myeloperoxidase,hematoxylin-eosin (H&E) straining, and immunostaining for neutrophilelastase to identify neutrophils. Neutrophil infiltration measured byMPO was decreased markedly in the cornea 24 hours after injury byinjection with clordronate-encapsulated liposome, compared toPBS-encapsulated liposome-injected controls (PBS-LIP). Infiltration ofinflammatory cells and neutrophils also was decreased markedly in themacrophage-depleted cornea.

FIG. 35. TSG-6 suppressed HSPB4-induced activation in macrophages in aCD44-dependant manner. A, B. TSG-6 in a dose-dependent manner suppressedthe activation of macrophages by HSPB4. C, D. TSG-6 did not inhibitHSPB4-mediated activation of NF-kB signaling in HEK-TLR2 cells which didnot express CD44. However, after the cells were transfected to expressCD44, TSG-6 dose-dependently inhibited HSPB4-mediated activation ofNF-kB signaling. E. TSG-6 inhibited significantly the inflammation inthe corneas of wild-type C57BL/6 mice, but it did not suppressinflammation in the corneas of CD44 knockout mice. F. Murine macrophages(RAW 264.7) were incubated with secretoneurin and evaluated forexpression of pro-inflammatory cytokines by real time RT-PCR.Secretoneurin did not activate macrophages. Human keratocytes werecultured with either secretoneurin or HSPB4. Neither secretoneurin norHSPB4 activated keratocytes to produce cytokines.

FIG. 36. The schematic diagram of sterile inflammation in the corneaImmediately after injury, SN is released from nerve endings in thecornea and circulating neutrophils are recruited, thereby inducing thePhase I inflammatory response. In response to injury including oxidativestress, necrotic or injured keratocytes secrete HSPB4. The HSPB4activates resident macrophages in the cornea via the TLR2/NF-kBsignaling pathway to produce pro-inflammatory cytokines including IL-1and IL-6. These injury signals are propagated rapidly and amplified bykeratocyte activation to produce chemokines that induce neutrophilinfiltration of Phase II. TSG-6 decreased neutrophil infiltration byinhibiting the initial step of macrophage activation via TLR2/CD44/NF-kBsignaling.

FIG. 37. Intravitreal administration of STC-1 (SEQ ID NO:2, whichcomprises C-Terminal Flag-tag) rescued photoreceptors in the S334ter-3rat. (A) Plot of ONL thickness taken from a total of 54 measurements ineach retina (27 superior retina and 27 inferior retina) from arepresentative animal demonstrated that STC-1 injected twicesignificantly improved outer nuclear layer (ONL) thickness compared touninjected (UI) controls. (B) Electroretinographic analysis performed atP19 following injection of STC-1 in the S334ter-3 rat at P9 (n=10).Response amplitudes are from a stimulus intensity of 0.4 log cd sec/m²in dark-adapted rats (scotopic b-wave) and light-adapted rats (photopicb-wave). Significant rescue of both scotopic and photopic b-waves wasobserved. (C-D) Light micrographs of the posterior retina of anS334ter-3 rat injected twice (at P9 and P12) with STC-1 and eyes takenat P19. The ONL in the treated eye (D) is approximately twice thethickness of that in the control eye (C), i.e., it containsapproximately twice the number of photoreceptors. RPE, retinal pigmentepithelium. Bar=20 nm. *P<0.05; **P<0.01. Error bars representmeans±s.e.m.

FIG. 38. Intravitreal administration of STC-1 (SEQ ID NO:2) rescuedphotoreceptor gene expression in the S334ter-3 rat. Real time RT-PCRanalysis for the photoreceptor genes. (A) Photoreceptor gene expressiondeclined rapidly from P12-P14. (B) Treatment with STC-1 rescuedexpression of four photoreceptor genes compared to uninjected (UI)control eyes from the same rats after one injection (1×STC-1) or twoinjections (2×STC-1). *P<0.05; **P<0.01. Error bars representmeans±s.e.m.

FIG. 39. Intravitreal administration of STC-1 (SEQ ID NO:2) rescuedphotoreceptors in the RCS rat. (A-B) Light micrographs of the posteriorretina of an RCS rat injected twice (at P21 and P28) with STC-1 and eyestaken at P42. The outer nuclear layer (ONL) in the two eyes is similarin thickness. However, in the uninjected control eye (A), a largepercentage of photoreceptor nuclei are dead and pyknotic (arrowheads),and some are coalesced into large masses of chromatin (arrows), typicalof RCS retinas at this age. In the STC-1 treated eye (B), far fewerpyknotic nuclei are present and the rod outer segment debris zone (D) isthicker than in the control eye. IS, photoreceptor inner segments; RPE,retinal pigment epithelium. Bar=20 μm. (C) Electroretinographic analysisperformed at P42 following injections of STC-1 in the RCS rat at P21 andP42. Response amplitudes are from a stimulus intensity of 0.4 log cdsec/m² in dark-adapted rats (scotopic b-wave). Significant rescue ofscotopic b-waves was observed. *P<0.05. Error bars representmeans±s.e.m.

FIG. 40. Intravitreal administration of STC-1 rescued photoreceptor geneexpression in the RCS rat. Real time RT-PCR analysis for thephotoreceptor genes. (A) Photoreceptor gene expression underwent agradual decline in the RCS rat between P21 and P45. The decline was moregradual than in the S334ter-3 rat (compare with FIG. 2). (B) Twoinjections of STC-1 (2×STC-1) rescued expression of the fourphotoreceptor genes compared to vehicle controls. A single injection ofSTC-1 (1×STC-1) increased the levels of three of the photoreceptortranscripts. *P<0.05; **P<0.01.

FIG. 41. Intravitreal administration of STC-1 (SEQ ID NO:2) increasedUCP-2 gene expression and decreased levels of ROS products in the RCSrat. (A) Real time RT-PCR analysis for the mitochondrial uncouplingprotein UCP-2 three days after injection of STC-1 at P21. (B, C) ELISAanalysis for two markers of oxidative damage in the retina: proteincarbonyl (B) and nitrotyrosine (C) content at P42 after injections ofSTC-1 at P21 and P28. Error bars represent means±s.e.m. *P<0.05;**P<0.01; ***P<0.001.

FIG. 42. hMSCs remain viable in the rat vitreous cavity and increaseexpression of STC-1. (A) Standard curve of real time RT-PCR assays forhuman GAPDH mRNA as a reflection of viable MSCs. Varying numbers ofhuman MSCs were added to whole globes just before RNA was extracted. (B)Recovery of viable human cells 5 days and 21 days after injection of100,000 human MSCs into the vitreous cavity of RCS rats. (C) Real timeRT-PCR assays of STC-1 expression in hMSCs prior to injections(Uninjected hMSCs) and in hMSCs recovered from globes 5 days afterinjection. Values were normalized by assays of human GAPDH mRNA fornumber of hMSCs recovered and calibrated to uninjected hMSCs (RQ).

FIG. 43. Rescue of mRNAs for photoreceptors by intravitreal injection ofhMSCs in RCS rats. Real time RT-PCR analysis for the photoreceptorgenes: rhodopsin, phosducin, neural retina leucine zipper, andrecoverin. Injection of hMSCs on P21 rescued expression of the genes atP42. *P<0.05.

DETAILED DESCRIPTION

The present invention in part relates to the finding that certainanti-apoptotic or anti-inflammatory polypeptides have application astherapies in the treatment of eye disease. For example, the inventorshave found that STC-1 is useful in the treatment of eye disease.Accordingly, these polypeptides and analogues thereof are useful astherapeutic agents in ocular diseases, including but not limited todiseases of the cornea and retina.

A. DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

As used herein, the term “biocompatible lattice,” is meant to refer to asubstrate that can facilitate formation into three-dimensionalstructures conducive for tissue development. Thus, for example, cellscan be cultured or seeded onto such a biocompatible lattice, such as onethat includes extracellular matrix material, synthetic polymers,cytokines, growth factors, etc. The lattice can be molded into desiredshapes for facilitating the development of tissue types. Also, at leastat an early stage during culturing of the cells, the medium and/orsubstrate is supplemented with factors (e.g., growth factors, cytokines,extracellular matrix material, etc.) that facilitate the development ofappropriate tissue types and structures.

As used herein, the term “bone marrow stromal cells,” “stromal cells,”“mesenchymal stem cells,” “mesenchymal stromal cells” or “MSCs” are usedinterchangeably and refer to a cell derived from bone marrow (reviewedin Prockop, 1997), peripheral blood (Kuznetsov et al., 2001), adiposetissue (Guilak et al., 2004), umbilical cord blood (Rosada et al.,2003), synovial membranes (De Bari et al., 2001), and periodontalligament (Seo et al., 2005), embryonic yolk sac, placenta, umbilicalcord, skin, and blood (U.S. Pat. Nos. 5,486,359 and 7,153,500), fat, andsynovial fluid. MSCs are characterized by their ability to adhere toplastic tissue culture surfaces (Friedenstein et al.; reviewed in Owen &Friedenstein, 1988), and by being effective feeder layers forhematopoietic stem cells (Eaves et al., 2001). In addition, MSCs can bedifferentiated both in culture and in vivo into osteoblasts andchondrocytes, into adipocytes, muscle cells (Wakitani et al., 1995) andcardiomyocytes (Fukuda and Yuasa, 2006), into neural precursors(Woodbury et al., 2000; Deng et al., 2001, Kim et al., 2006; Marcsehi etal., 2006; Krampera et al., 2007). Mesenchymal stem cells (MSCs) may bepurified using methods known in the art (Wakitani et al., 1995; Fukudaand Yuasa, 2006; Woodbury et al., 2000; Deng et al., 2001; Kim et al.,2006; Mareschi et al., 2006; Krampera et al., 2007), and serve asprogenitors for mesenchymal cell lineages, including bone, cartilage,ligament, tendon, adipose, muscle, cardiac tissue, stroma, dermis, andother connective tissues. (See U.S. Pat. Nos. 6,387,369 and 7,101,704).

As used herein, the term “modulate” is meant to refer to any change inbiological state, including increasing, decreasing, and the like.

The terms “precursor cell,” “progenitor cell,” and “stem cell” are usedinterchangeably in the art and herein and refer either to a pluripotent,or lineage-uncommitted, progenitor cell, which is potentially capable ofan unlimited number of mitotic divisions to either renew itself or toproduce progeny cells which will differentiate into the desired celltype. Unlike pluripotent stem cells, lineage-committed progenitor cellsare generally considered to be incapable of giving rise to numerous celltypes that phenotypically differ from each other. Instead, progenitorcells give rise to one or possibly two lineage-committed cell types.

“Ocular region” or “ocular site” means any area of the ocular globe(eyeball), including the anterior and posterior segment of the eye, andwhich generally includes, but is not limited to, any functional (e.g.,for vision) or structural tissues found in the eyeball, or tissues orcellular layers that partly or completely line the interior or exteriorof the eyeball. Specific examples of areas of the eyeball in an ocularregion include, but are not limited to, the anterior chamber, theposterior chamber, the vitreous cavity, the choroid, the suprachoroidalspace, the conjunctiva, the subconjunctival space, the episcieral space,the intracorneal space, the subretinal space, sub-Tenon's space, theepicorneal space, the sclera, the pars plana, surgically-inducedavascular regions, the macula, and the retina.

“Ocular condition” means a disease, ailment or condition which affectsor involves the eye or one of the parts or regions of the eye. Broadlyspeaking, the eye includes the eyeball, including the cornea, and othertissues and fluids which constitute the eyeball, the periocular muscles(such as the oblique and rectus muscles) and the portion of the opticnerve which is within or adjacent to the eyeball.

“Graft” refers to a cell, tissue, organ, or otherwise any biologicalcompatible lattice for transplantation.

“Glaucoma” means primary, secondary and/or congenital glaucoma. Primaryglaucoma can include open angle and closed angle glaucoma. Secondaryglaucoma can occur as a complication of a variety of other conditions,such as injury, inflammation, pigment dispersion, vascular disease anddiabetes. The increased pressure of glaucoma causes blindness because itdamages the optic nerve where it enters the eye. Thus, in onenon-limiting embodiment, by lowering reactive oxygen species, STC-1, orMSCs which express increased amounts of STC-1, may be employed in thetreatment of glaucoma and prevent or delay the onset of blindness.

“Inflammation-mediated” in relation to an ocular condition means anycondition of the eye which can benefit from treatment with ananti-inflammatory agent, and is meant to include, but is not limited to,uveitis, macular edema, acute macular degeneration, retinal detachment,ocular tumors, fungal or viral infections, multifocal choroiditis,diabetic retinopathy, uveitis, proliferative vitreoretinopathy (PVR),sympathetic ophthalmia, Vogt-Koyanagi-Harada (VKH) syndrome,histoplasmosis, and uveal diffusion.

“Injury” or “damage” are interchangeable and refer to the cellular andmorphological manifestations and symptoms resulting from aninflammatory-mediated condition, such as, for example, inflammation, aswell as tissue injuries caused by means other than inflammation, such aschemical injury, including chemical burns, as well as injuries caused byinfections, including but not limited to, bacterial, viral, or fungalinfections.

“Intraocular” means within or under an ocular tissue. An intraocularadministration of a drug delivery system includes administration of thedrug delivery system to a sub-tenon, subconjunctival, suprachoroidal,subretinal, intravitreal, anterior chamber, and the like location. Anintraocular administration of a drug delivery system excludesadministration of the drug delivery system to a topical, systemic,intramuscular, subcutaneous, intraperitoneal, and the like location.

“Macular degeneration” refers to any of a number of disorders andconditions in which the macula degenerates or loses functional activity.The degeneration or loss of functional activity can arise as a resultof, for example, cell death, decreased cell proliferation, loss ofnormal biological function, or a combination of the foregoing. Maculardegeneration can lead to and/or manifest as alterations in thestructural integrity of the cells and/or extracellular matrix of themacula, alteration in normal cellular and/or extracellular matrixarchitecture, and/or the loss of function of macular cells. The cellscan be any cell type normally present in or near the macula includingRPE cells, photoreceptors, and capillary endothelial cells. Age-relatedmacular degeneration, or ARMD, is the major macular degeneration relatedcondition, but a number of others are known including, but not limitedto, Best macular dystrophy, Stargardt macular dystrophy, Sorsby fundusdystrophy, Mallatia Leventinese, Doyne honeycomb retinal dystrophy, andRPE pattern dystrophies. Age-related macular degeneration (AMD) isdescribed as either “dry” or “wet.” The wet, exudative, neovascular formof AMD affects about 10-20% of those with AMD and is characterized byabnormal blood vessels growing under or through the retinal pigmentepithelium (RPE), resulting in hemorrhage, exudation, scarring, orserous retinal detachment. Eighty to ninety percent of AMD patients havethe dry form characterized by atrophy of the retinal pigment epitheliumand loss of macular photoreceptors. Drusen may or may not be present inthe macula. There may also be geographic atrophy of retinal pigmentepithelium in the macula accounting for vision loss. At present there isno cure for any form of AMD, although some success in attenuation of wetAMD has been obtained with photodynamic and especially anti-VEGFtherapy.

“Drusen” is debris-like material that accumulates with age below theRPE. Drusen is observed using a funduscopic eye examination. Normal eyesmay have maculas free of drusen, yet drusen may be abundant in theretinal periphery. The presence of soft drusen in the macula, in theabsence of any loss of macular vision, is considered an early stage ofAMD. Drusen contains a variety of lipids, polysaccharides, andglycosaminoglycans along with several proteins, modified proteins orprotein adducts. There is no generally accepted therapeutic method thataddresses drusen formation and thereby manages the progressive nature ofAMD.

“Ocular neovascularization” (ONV) is used herein to refer to choroidalneovascularization or retinal neovascularization, or both.

“Retinal neovascularization” (RNV) refers to the abnormal development,proliferation, and/or growth of retinal blood vessels, e.g., on theretinal surface.

“Subretinal neovascularization” (SRNVM) refers to the abnormaldevelopment, proliferation, and/or growth of blood vessels beneath thesurface of the retina.

“Cornea” refers to the transparent structure forming the anterior partof the fibrous tunic of the eye. It consists of five layers,specifically: 1) anterior corneal epithelium, continuous with theconjunctiva; 2) anterior limiting layer (Bowman's layer); 3) substantiapropria, or stromal layer; 4) posterior limiting layer (Descemet'smembrane); and 5) endothelium of the anterior chamber or keratoderma.

“Retina” refers to the innermost layer of the ocular globe surroundingthe vitreous body and continuous posteriorly with the optic nerve. Theretina is composed of layers including the: 1) internal limitingmembrane; 2) nerve fiber layer; 3) layer of ganglion cells; 4) innerplexiform layer; 5) inner nuclear layer; 6) outer plexiform layer; 7)outer nuclear layer; 8) external limiting membrane; and 9) a layer ofrods and cones.

“Retinal degeneration” refers to any hereditary or acquired degenerationof the retina and/or retinal pigment epithelium. Non-limiting examplesinclude retinitis pigmentosa, Best's Disease, RPE pattern dystrophies,and age-related macular degeneration.

“Allogeneic” refers to a graft derived from a different animal of thesame species. As defined herein, an “allogeneic bone marrow stromal cell(BMSC)” is obtained from a different individual of the same species asthe recipient.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

“Transplant” refers to a biocompatible lattice or a donor tissue, organor cell, to be transplanted. An example of a transplant may include butis not limited to skin cells or tissue, bone marrow, and solid organssuch as heart, pancreas, kidney, lung and liver. Preferably, thetransplant is a human neural stem cell.

“Donor antigen” refers to an antigen expressed by the donor tissue to betransplanted into the recipient.

“Alloantigen” is an antigen that differs from an antigen expressed bythe recipient.

As used herein, a “therapeutically effective amount” is the amount of anagent which is sufficient to provide a beneficial effect to the subjectto which the agent is administered.

As used herein “endogenous” refers to any material from or producedinside an organism, cell or system.

“Exogenous” refers to any material introduced from or produced outsidean organism, cell, or system.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

An “isolated polypeptide” refers to a polypeptide that has beensubstantially purified or separated from other components whichnaturally accompany it.

A “polypeptide” as used herein refers to a consecutive series of 5 ormore amino acid residues. As used herein, a “peptide,” and a “protein”are examples of polypeptides so long as they include a consecutiveseries of 5 or more amino acid residues.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnonviral compounds which facilitate transfer of nucleic acid into cells,such as, for example, polylysine compounds, liposomes, and the like.Examples of viral vectors include, but are not limited to, adenoviralvectors, adeno-associated virus vectors, retroviral vectors, and thelike.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

B. THERAPEUTIC POLYPEPTIDES

The present invention pertains to use of therapeutic polypeptides invarious contexts. For example, some aspects of the present inventionpertain to methods for treating an eye disease comprising contacting theeye of a subject with a STC-1 polypeptide, a STC-2 polypeptide, or aTSG-6 polypeptide. In some aspects, the subject is administered acomposition comprising cells that overexpress a STC-1 polypeptide, aSTC-2 polypeptide, or a TSG-6 polypeptide.

1. Stanniocalcin 1

The stanniocalcin (STC) family of proteins includes stanniocalcin 1(STC-1), stanniocalcin 2 (STC-2), and is defined herein to further referto molecules that have at least 30% sequence identity to a naturallyoccurring STC-1 or STC-2 protein and have at least one common biologicalfunction of a naturally occurring STC-1 or STC-2 protein. STC-1 is amammalian protein that has been implicated to play an autocrine andparacrine role in mammals, leading to various effects. It is expressedin many tissues, but is not normally detected in the circulation. Inmice, it has been shown to affect calcium homeostasis, bone and musclestructure, and angiogenesis through effects of a variety of cellsincluding osteoblasts, osteoclasts, myocytes, and endothelial cells.STC-1 has also been implicated to be involved in calcium homeostasis inthe heart. STC-1 has also been shown to be secreted by mesenchymal stemcells (MSCs) that have been exposed to injured fibroblasts (Block etal., 2009). Some examples of STC-1 polypeptides are set forth in Table 1below. In each case where an amino acid or polynucleotide sequencereferenced herein is attributed to a database accession number, theamino acid and/or polynucleotide sequence archived in the database forthe accession number is incorporated herein by reference as thatsequence is presented in the database on the filing date of thisspecification. Each sequence attributed to an accession number recitedherein can be readily accessed via, for example, the National Center forBiotechnology (NCBI) database, which is available online to the public.

TABLE 1 STC-1 Proteins and Precursors SEQ ID NO: Description SpeciesAdditional Information 1 240 AA, STC-1, Human, source HEK293 2 240 AA,STC-1 Human; source− with C-terminal HEK293 Flag tag (10 AA) 3 247 AAHuman Accession: AAL79522.1 4 247 AA Human Accession: EAW63610.1 5 247aa, STC-1 Human Accession: precursor NP_003146.1 6 247 aa, STC-1Synthetic Accession: construct ABM84894.1 7 247 aa, STC-1 SyntheticAccession: construct ABM81739.1 8 247 aa, STC-1 Mus musculus Accession:precursor NP_033311.3 9 247 AA, STC-1 Rattus Accession: precursornorvegicus NP_112385.1 10 247 AA, STC-1 Rattus Accession: norvegicusEDM02180.1 11 246 AA, STC-1 Mus musculus Accession: AAP47156.1

2. Stanniocalcin 2

Stanniocalcin 2 (STC-2) is related to STC-1. It is expressed in a widevariety of tissues. In the ovary of certain mammals, it has been shownto be a paracrine hormone that regulates granulosa cell formation. STC-2has also been shown to be upregulated in neuronal cells by oxidativestress and hypoxia. Induced STC-2 expression has been shown to be anessential feature of the survival component of the unfolded proteinresponse (UPR) (Ito et al., Mol. Cell. Biol. 24 (21):9456-9469). Someexamples of STC-2 polypeptides are set forth in Table 2 below.

TABLE 2 STC-2 Proteins and Precursors Additional SEQ ID NO: DescriptionSpecies Information 12 STC-2, 302 aa Human Accession: AAV38398.1 13STC-2, 302 aa Human Accession: AAH13958.1 14 STC-2, 302 aa HumanAccession: AAH06352.1 15 STC-2, 302 aa Human Accession: AAH00658.1 16STC-2 precursor, Human Accession: 302 aa NP_003705.1 17 STC-2, 296 aaMus musculus Accession: EDL23779.1 18 STC-2, 296 aa Mus musculusAccession: AAH12206.1 19 STC-2, 296 aa Rattus Accession: norvegicusEDM04038.1 20 STC-2 precursor, Rattus Accession: 296 aa norvegicusNP_071566.1

3. Tumor Necrosis Factor-Inducible Gene 6 Protein

Tumor Necrosis Factor-Inducible Gene 6 Protein, also known asTNF-stimulated gene 6 protein or TSG-6, is a 30 kDa secreted proteinthat is a member of the hyaluronan-binding protein family. It contains ahyaluronan-binding LINK domain. TSG-6 is involved in extracellularmatrix stability and cell migration. Expression of TSG-6 is induced bysignaling molecules such as tumor necrosis factor α (TGF-α) andinterleukin 2 (IL-2). Expression of TSG-6 is also correlated withproteoglycan synthesis in vascular smooth muscle cells. Some examples ofTSG-6 polypeptides are set forth in Table 3 below.

TABLE 3 TSG-6 Proteins and Precursors Additional SEQ ID NO: DescriptionSpecies Information 21 TSG-6, 277 aa Human Accession: P98066 22 TSG-6,277 aa Human Accession CAD13434.1 23 TSG-6, 277 aa Human Accession:CAD12353.1 24 TSG-6 precursor, Human Accession: 277 aa NP_009046.2 25TSG-6 precursor, Mus musculus Accession: 275 aa NP_033424.1

Throughout this application, the term “therapeutic polypeptide” isintended to refer to a polypeptide that has a therapeutic effect on aneye disease in a mammalian subject.

It is well understood by the skilled artisan that, inherent in thedefinition of a “therapeutic polypeptide” (including therapeutic STC-1polypeptide, STC-2 polypeptide, and TSG-6 polypeptide) is the conceptthat there is a limit to the number of changes that may be made within adefined portion of the molecule and still result in a molecule with anacceptable level of equivalent biological activity, e.g., ability of thepolypeptide to retain at least 95% of the biological activity of thenaturally occurring protein sequence (including any of the full-lengthprotein sequences set forth in Tables 1, 2, or 3). “Therapeuticpolypeptide” is thus defined herein as any therapeutic polypeptide inwhich some, or most, of the amino acids may be substituted so long asthe polypeptide retains substantially similar activity in the context ofthe uses set forth herein.

An amino acid sequence of any length is contemplated within thedefinition of therapeutic polypeptide so long as the polypeptide retainsan acceptable level of equivalent biological activity of the nativesequence. For example, a STC-1 polypeptide, a STC-2 polypeptide, or aTSG-6 polypeptide includes homologs, variants, and fragments of thenative sequences of STC-1, STC-2, and TSG-6 so long as the polypeptideretains an acceptable level of equivalent biological activity as thenative sequence of STC-1, STC-2, or TSG-6, respectively. Biologicalactivities of STC-1, STC-2, and TSG-6 are set forth in this section.Variants of therapeutic polypeptides will preferably have at least 80%,82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or greater sequenceidentity to the native sequence of the therapeutic polypeptide. Examplesof a native sequence of STC-1 include SEQ ID NOs: 1, 3, or 4. Examplesof a native sequence of STC-2 include SEQ ID NOs: 12, 13, 14, and 15.Examples of a native sequence of TSG-6 include SEQ ID NOs: 21, 22, and23. A “native sequence” polypeptide comprises a polypeptide having thesame amino acid sequence as the corresponding polypeptide derived fromnature.

“Percent (%) amino acid sequence identity” or “homology” with respect toa polypeptide sequence identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the polypeptide being compared, after aligningthe sequences considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared.

The present invention may utilize therapeutic polypeptides purified froma natural source or from recombinantly-produced material. Those ofordinary skill in the art would know how to produce these polypeptidesfrom recombinantly-produced material. This material may use the 20common amino acids in naturally synthesized proteins, or one or moremodified or unusual amino acids. Generally, “purified” will refer to atherapeutic polypeptide composition that has been subjected tofractionation to remove various other proteins, polypeptides, orpeptides, and which composition substantially retains its activity.“Purified” and “isolated” are used interchangeably herein. Purificationmay be substantial, in which the therapeutic polypeptide is thepredominant species, or to homogeneity, which purification level wouldpermit accurate degradative sequencing. An “isolated polypeptide”includes a polypeptide that has been separated from a cell that producedthe polypeptide where the polypeptide is purified from a natural source.

Therapeutic polypeptides may be amino acid sequence mutants of thenaturally occurring polypeptide sequence. Amino acid sequence mutants ofthe polypeptide can be substitutional mutants or insertional mutants.Insertional mutants typically involve the addition of material at anon-terminal point in the peptide. This may include the insertion of afew residues, an immunoreactive epitope, or simply a single residue. Theadded material may be modified, such as by methylation, acetylation, andthe like. Alternatively, additional residues may be added to theN-terminal or C-terminal ends of the peptide. Amino acid substitutionsare generally based on the relative similarity of the amino acidside-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, and the like. An analysis of the size,shape and type of the amino acid side-chain substituents reveals thatarginine, lysine and histidine are all positively charged residues; thatalanine, glycine and serine are all a similar size; and thatphenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

C. THERAPEUTIC NUCLEIC ACIDS

Various aspects of the present invention require polynucleotidesencoding any of the foregoing therapeutic polypeptides. For example,various embodiments include methods for treating an eye disease thatinvolve contacting the eye with an expression cassette that includes apromoter that is active in a cell of the mammalian eye, operably linkedto a polynucleotide encoding either a therapeutic polypeptide as setforth herein.

1. Polynucleotides Generally

In some embodiments, the polynucleotides may be derived from genomic DNAor may be complementary DNA (cDNA). cDNA is DNA prepared using messengerRNA (mRNA) as a template. Thus, a cDNA does not contain any interruptedcoding sequences and usually contains almost exclusively the codingregion(s) for the corresponding protein. In other embodiments, thepolynucleotide may be produced synthetically.

It may be advantageous to combine portions of the genomic DNA with cDNAor synthetic sequences to generate specific constructs. For example,where an intron is desired in the ultimate construct, a genomic clonewill need to be used. Introns may be derived from other genes inaddition to the gene encoding the therapeutic polypeptide. The cDNA or asynthesized polynucleotide may provide more convenient restriction sitesfor the remaining portion of the construct and, therefore, would be usedfor the rest of the sequence.

In certain embodiments, one may wish to employ constructs which includeother elements, for example, those which include C-5 propynepyrimidines. Oligonucleotides which contain C-5 propyne analogues ofuridine and cytidine have been shown to bind RNA with high affinity(Wagner et al., 1993).

2. Expression Cassettes

Certain embodiments of the invention pertain to methods utilizingcompositions that include an expression cassette. In particular, themethods for treating eye disease may involve administering a therapeuticpolynucleotide that is comprised in an expression cassette.

Throughout this application, the term “expression cassette” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein or polypeptide, but it need not be. Thus, incertain embodiments, expression includes both transcription of a geneand translation of a mRNA into a polypeptide.

In order for the expression cassette to effect expression of atherapeutic polypeptide, the polynucleotide encoding the polynucleotidewill be under the transcriptional control of a promoter. A “promoter” isa control sequence that is a region of a nucleic acid sequence at whichinitiation and rate of transcription are controlled. It may containgenetic elements at which regulatory proteins and molecules may bindsuch as RNA polymerase and other transcription factors. The phrase“operatively linked” means that a promoter is in a correct functionallocation and/or orientation in relation to a nucleic acid sequence tocontrol transcriptional initiation and/or expression of that sequence. Apromoter may or may not be used in conjunction with an “enhancer,” whichrefers to a cis-acting regulatory sequence involved in thetranscriptional activation of a nucleic acid sequence. One of skill inthe art would understand how to use a promoter or enhancer to promoteexpression of therapeutic polynucleotide.

In certain embodiments of the invention, the delivery of an expressioncassette in a cell may be identified in vitro or in vivo by including amarker in the expression vector. The marker would result in anidentifiable change to the transfected cell permitting easyidentification of expression. The selectable marker employed is notbelieved to be important, so long as it is capable of being expressedalong with the polynucleotide of the expression cassette. Examples ofselectable markers are well known to one of skill in the art.

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). One ofskill in the art would be familiar with use of IRES in expressioncassettes. Expression cassettes can include a multiple cloning site(MCS), which is a nucleic acid region that contains multiple restrictionenzyme sites, any of which can be used in conjunction with standardrecombinant technology to digest the vector. See Carbonelli et al.(1999); Levenson et al. (1998); Cocea (1997). “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Techniques involving restriction enzymes and ligationreactions are well known to those of skill in the art of recombinanttechnology.

In expression, one will typically include a polyadenylation signal toeffect proper polyadenylation of the transcript. The nature of thepolyadenylation signal is not believed to be crucial to the successfulpractice of the invention, and/or any such sequence may be employed. Oneof skill in the art would understand how to use these signals to effectproper polyadenylation of the transcript.

In certain embodiments of the present invention, the expression cassettecomprises a virus or engineered construct derived from a viral genome.The ability of certain viruses to enter cells via receptor-mediatedendocytosis and, in some cases, integrate into the host cellchromosomes, have made them attractive candidates for gene transfer into mammalian cells. However, because it has been demonstrated thatdirect uptake of naked DNA, as well as receptor-mediated uptake of DNAcomplexes, is possible, expression vectors need not be viral but,instead, may be any plasmid, cosmid or phage construct that is capableof supporting expression of encoded genes in mammalian cells, such aspUC or Bluescript™ plasmid series. One of ordinary skill in the artwould be familiar with use of viruses as tools to promote expression ofthe polypeptide.

In certain embodiments of the invention, a treated cell may beidentified in vitro or in vivo by including a marker in the expressionvector. Such markers would confer an identifiable change to the cellpermitting easy identification of cells containing the expressionvector. Generally, a selectable marker is one that confers a propertythat allows for selection. A positive selectable marker is one in whichthe presence of the marker allows for its selection, while a negativeselectable marker is one in which its presence prevents its selection.An example of a positive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

3. Viral Vectors

In certain embodiments, the methods and compositions of the inventionutilize expression cassettes which includes the therapeutic polypeptidein an expression cassette carried in a vector. One of ordinary skill inthe art would understand use of vectors since these experimental methodsare well-known in the art. In particular, techniques using “viralvectors” are well-known in the art. A viral vector is meant to includethose constructs containing viral sequences sufficient to (a) supportpackaging of the expression cassette and (b) to ultimately express arecombinant gene construct that has been cloned therein.

One method for delivery of the recombinant DNA involves the use of anadenovirus expression vector. Although adenovirus vectors are known tohave a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. Adenoviruses are currently the most commonly used vectorfor gene transfer in clinical settings. Among the advantages of theseviruses is that they are efficient at gene delivery to both nondividingand dividing cells and can be produced in large quantities. The vectorcomprises a genetically engineered form of adenovirus. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double-stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus et al., 1992). In contrast toretrovirus, the adenoviral infection of host cells does not result inchromosomal integration because adenoviral DNA can replicate in anepisomal manner without potential genotoxicity. Also, adenoviruses arestructurally stable, and no genome rearrangement has been detected afterextensive amplification. Adenovirus is particularly suitable for use asa gene transfer vector because of its mid-sized genome, ease ofmanipulation, high titer, wide target-cell range and high infectivity. Aperson of ordinary skill in the art would be familiar with experimentalmethods using adenoviral vectors.

Other types of viral vectors contemplated for use in the presentinvention include Adeno-associated virus (AAV), lentivirus, Herpessimplex virus (HSV), and Vaccinia virus.

4. Nonviral Vectors

Several non-viral methods for the transfer of expression vectors intocells also are contemplated by the present invention. These includecalcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen andOkayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985),electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), directmicroinjection (Harland and Weintraub, 1985), DNA-loaded liposomes(Nicolau and Sene, 1982; Fraley et al., 1979) and Lipofectamine-DNAcomplex, cell sonication (Fechheimer et al., 1987), gene bombardmentusing high velocity microprojectiles (Yang et al., 1990), polycations(Bousssif et al., 1995) and receptor-mediated transfection (Wu and Wu,1987; Wu and Wu, 1988). Some of these techniques may be successfullyadapted for in vivo or ex vivo use. A person of ordinary skill in theart would be familiar with the techniques pertaining to use of nonviralvectors, and would understand that other types of nonviral vectors thanthose disclosed herein are contemplated by the present invention.

In a further embodiment of the invention, the expression cassette may beentrapped in a liposome or lipid formulation. Liposomes are vesicularstructures characterized by a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution.

Nanoparticles, such as chitosan nanoparticles are also contemplated asnonviral vectors for use in the context of the present invention.

D. TARGETED DISEASES AND CONDITIONS

The present invention contemplates methods of treating a subject with aneye disease or condition that includes administering to the subject acomposition that includes a therapeutic polypeptide as set forth hereinin a pharmaceutical preparation suitable for delivery to the subject.The eye disease may be any disease in which apoptosis or inflammationhas been implicated to play a role in the pathophysiology. Non-limitingexamples are set forth below.

1. Ocular injuries

a. Foreign Bodies

25% of all ocular injuries involve foreign bodies on the surface of thecornea. No scarring will occur if the injury affects only the cornealepithelium; but if it also affects the Bowman zone, scarring ispossible. After removal of the foreign body, the eye is treated with asulfonamide or antibiotic and, if there is ciliary congestion andphotophobia, or if the removal of the foreign body were difficult, it istreated with a cycloplegic such as 5% homatropine. In some instances,the therapeutic compositions of the present invention are designed toaccelerate healing of the injury caused by the foreign body and toprevent infection, and to improve the clinical outcome.

b. Chemical Burns

Chemical burns are treated by first diluting the chemical by flushingthe eye with fluid, and then preventing infection through the use oftopical antibiotics.

Intraocular pressure may be reduced by applying timolol, epinephrine,acetazolamide, or other similar agents. If epithelialization of thecornea is incomplete after one week, there is a danger of stromalnecrosis, in addition to the risk of infection. It is therefore criticalthat the healing be accelerated to reduce these risks.

Severe scarring is another common result of chemical burns. Thetherapeutic compositions of the present invention are designed toaccelerate the healing of the corneal erosion caused by the chemicalburns, to prevent stromal necrosis and infection of the eye, and toreduce corneal scarring and thereby restore/preserve cornealtransparency.

Unexpectedly the compositions of the present invention are able toprevent or reduce scar formation while simultaneously enhancing ocularhealing, wound repair, and maintaining corneal transparency. While notwishing to be bound by any specific mechanism of action, it appears thatthese beneficial effects can be obtained due to the anti-inflammatoryactions of the compositions. In some instances, the beneficial effectscan be obtained due to the combination of anti-inflammatory andanti-apoptotic actions of the compositions of the invention.

c. Lacerations

Lacerations of the cornea are followed by prolapse of the iris, whichcloses the injury. As in all eye injuries, there is a risk of infection.Lacerations also may extend to the sclera, which is a much more severeinjury. In such a case, surgery is required to remove prolapsed uvealtissue from the injured area, and the sclera is closed with sutures. Thetherapeutic compositions of the present invention are designed toaccelerate the healing of the laceration and to prevent infection.

2. Optic Neuropathies

Optic neuropathies affect the optic nerve, which may affect visionadversely. Traumatic optic neuropathies may be caused when the head isstruck by an object, such as a ball, or if it is pierced by an objectsuch as a bullet. Toxic optic neuropathies are caused by chemicals toxicto the optic nerve; a common example is the ingestion of methanol.Deficiency optic neuropathies can result from vitamin deficiencies suchas a B 12 deficiency and may cause lesions in the optic nerve.Hereditary optic neuropathies can be caused by mutations in the nuclearor mitochondrial genomes. The therapeutic and prophylactic compounds ofthis invention could be used to heal the optic nerve throughanti-inflammatory and anti-apoptotic mechanisms. They may also create anenvironment which would reduce or prevent mutations in optic cellgenomes.

3. Inflammatory Conditions

Inflammation-mediated conditions of the eye which may be treated by themethods of the invention include but are not limited to uveitis, macularedema, age-related macular degeneration, retinal detachment, oculartumors, fungal or viral infections, multifocal choroiditis, diabeticuveitis, proliferative vitreoretinopathy (PVR), sympathetic opthalmia,Vogt Koyanagi-Harada (VKH) syndrome, histoplasmosis, and uvealdiffusion. In a non-limiting embodiment, the inflammation-mediatedcondition of the eye is uveitis. In another non-limiting embodiment, theinflammation-mediated condition of the eye is proliferativevitreoretinopathy (PVR).

Suspensions of microspheres may be used as an anti-inflammatory therapyof the eye, especially for treating inflammatory conditions of theocular adnexa, palpebral or bulbar conjunctiva, cornea and anteriorsegment of the globe. Common therapeutic applications foranti-inflammatory suspensions of microspheres include viral, allergicconjunctivitis, acne rosacea, iritis and iridocyclitis. Microspheres mayalso be used to ameliorate inflammation associated with, corneal injurydue to chemical or thermal burns, or penetration of foreign bodies. Suchconditions may result from surgery, injury, allergy or infection to theeye and can cause severe discomfort.

Notably, microspheres have considerable therapeutic advantages inreducing inflammatory responses, compared to the prevalent topicalocular use of NSAI agents and corticosteroids. Use of topical steroidsis associated with a number of complications, including posteriorsubcapsular cataract formation, elevation of intraocular pressure,secondary ocular infection, retardation of corneal wound healing,uveitis, mydriasis, transient ocular discomfort and ptosis. Numeroussystemic complications also may arise from the topical ocularapplication of corticosteroids. These complications include adrenalinsufficiency, Cushing's syndrome, peptic ulceration, osteoporosis,hypertension, muscle weakness or atrophy, inhibition of growth,diabetes, activation of infection, mood changes and delayed woundhealing.

4. Ocular Surgical Applications

Compositions of microspheres in accordance with the present invention,may also be used to ameliorate inflammation associated with ocularsurgery, and in this context are particularly useful in a prophylacticmodality as well as in promoting healing and reducing scarring as hasbeen detailed above.

Of particular suitability is the use of the compositions of theinvention for: post trabeculectomy (filtering surgery); post pterygiumsurgery; post ocular adnexa trauma and surgery; post intraocular surgeryand specifically: post lensectomy, post vitrectomy, post retinaldetachment surgery, and post epi- and subretinal membrane peeling.

It will be appreciated by the artisan that these are intended to serveas non-limitative examples of prevalent surgical procedures for whichthe compositions and methods of the invention are useful.

5. Retinal Disease

The invention provides a method of preventing or treating various oculardiseases or conditions of the retina, including the following:maculopathies/retinal degeneration: macular degeneration, includingage-related macular degeneration (ARMD), such as non-exudativeage-related macular degeneration and exudative age-related maculardegeneration; choroidal neovascularization; retinopathy, includingdiabetic retinopathy, acute and chronic macular neuroretinopathy,central serous chorioretinopathy; and macular edema, including cystoidmacular edema, and diabetic macular edema.Uveitis/retinitis/choroiditis: acute multifocal placoid pigmentepitheliopathy, Behcet's disease, birdshot retinochoroidopathy,infectious (syphilis, Lyme Disease, tuberculosis, toxoplasmosis),uveitis, including intermediate uveitis (pars planitis) and anterioruveitis, multifocal choroiditis, multiple evanescent white dot syndrome(MEWDS), ocular sarcoidosis, posterior scleritis, serpignouschoroiditis, subretinal fibrosis, uveitis syndrome, andVogt-Koyanagi-Harada syndrome. Vascular diseases/exudative diseases:retinal arterial occlusive disease, central retinal vein occlusion,disseminated intravascular coagulopathy, branch retinal vein occlusion,hypertensive fundus changes, ocular ischemic syndrome, retinal arterialmicroaneurysms, Coats disease, parafoveal telangiectasis, hemi-retinalvein occlusion, papillophlebitis, central retinal artery occlusion,branch retinal artery occlusion, carotid artery disease (CAD), frostedbranch angitis, sickle cell retinopathy and other hemoglobinopathies,angioid streaks, familial exudative vitreoretinopathy, Eales disease,Traumatic/surgical diseases: sympathetic ophthalmia, uveitic retinaldisease, retinal detachment, trauma, laser, PDT, photocoagulation,hypoperfusion during surgery, radiation retinopathy, bone marrowtransplant retinopathy. Proliferative disorders: proliferative vitrealretinopathy and epiretinal membranes, proliferative diabeticretinopathy. Infectious disorders: ocular histoplasmosis, oculartoxocariasis, ocular histoplasmosis syndrome (OHS), endophthalmitis,toxoplasmosis, retinal diseases associated with HIV infection, choroidaldisease associated with HIV infection, uveitic disease associated withHIV Infection, viral retinitis, acute retinal necrosis, progressiveouter retinal necrosis, fungal retinal diseases, ocular syphilis, oculartuberculosis, diffuse unilateral subacute neuroretinitis, and myiasis.Genetic disorders: retinitis pigmentosa, systemic disorders withassociated retinal dystrophies, congenital stationary night blindness,cone dystrophies, Stargardt's disease and fundus flavimaculatus, Best'sdisease, pattern dystrophy of the retinal pigment epithelium, X-linkedretinoschisis, Sorsby's fundus dystrophy, benign concentric maculopathy,Bietti's crystalline dystrophy, pseudoxanthoma elasticum. Retinaltears/holes: retinal detachment, macular hole, giant retinal tear.Tumors: retinal disease associated with tumors, congenital hypertrophyof the RPE, posterior uveal melanoma, choroidal hemangioma, choroidalosteoma, choroidal metastasis, combined hamartoma of the retina andretinal pigment epithelium, retinoblastoma, vasoproliferative tumors ofthe ocular fundus, retinal astrocytoma, intraocular lymphoid tumors.Miscellaneous: punctate inner choroidopathy, acute posterior multifocalplacoid pigment epitheliopathy, myopic retinal degeneration, acuteretinal pigment epithelitis and the like.

5. Other Anterior Ocular Conditions

An anterior ocular condition is a disease, ailment or condition whichaffects or which involves an anterior (i.e., front of the eye) ocularregion or site, such as a periocular muscle, an eyelid or an eyeballtissue or fluid which is located anterior to the posterior wall of thelens capsule or ciliary muscles. Thus, an anterior ocular conditionprimarily affects or involves the conjunctiva, the cornea, the anteriorchamber, the iris, the posterior chamber (behind the iris but in frontof the posterior wall of the lens capsule), the lens or the lens capsuleand blood vessels and nerve which vascularize or innervate an anteriorocular region or site.

Thus, an anterior ocular condition can include a disease, ailment orcondition, such as for example, aphakia; pseudophakia; astigmatism;blepharospasm; cataract; conjunctival diseases; conjunctivitis,including, but not limited to, atopic keratoconjunctivitis; cornealinjuries, including, but not limited to, injury to the corneal stromalareas; corneal diseases; corneal ulcer; dry eye syndromes; eyeliddiseases; lacrimal apparatus diseases; lacrimal duct obstruction;myopia; presbyopia; pupil disorders; refractive disorders andstrabismus. Glaucoma can also be considered to be an anterior ocularcondition because a clinical goal of glaucoma treatment can be to reducea hypertension of aqueous fluid in the anterior chamber of the eye (i.e.reduce intraocular pressure).

Other diseases or disorders of the eye which may be treated inaccordance with the present invention include, but are not limited to,ocular cicatricial pemphigoid (OCP), Stevens Johnson syndrome andcataracts.

6. Other Ocular Conditions

A posterior ocular condition is a disease, ailment or condition whichprimarily affects or involves a posterior ocular region or site such aschoroid or sclera (in a position posterior to a plane through theposterior wall of the lens capsule), vitreous, vitreous chamber, retina,optic nerve (i.e., the optic disc), and blood vessels and nerves whichvascularize or innervate a posterior ocular region or site. Thus, aposterior ocular condition can include a disease, ailment or condition,such as for example, acute macular neuroretinopathy; Behcet's disease;choroidal neovascularization; diabetic retinopathy; uveitis; ocularhistoplasmosis; infections, such as fungal or viral-caused infections;macular degeneration, such as acute macular degeneration, non-exudativeage-related macular degeneration and exudative age-related maculardegeneration; edema, such as macular edema, cystoid macular edema anddiabetic macular edema; multifocal choroiditis; ocular trauma whichaffects a posterior ocular site or location; ocular tumors; retinaldisorders, such as central retinal vein occlusion, diabetic retinopathy(including proliferative diabetic retinopathy), proliferativevitreoretinopathy (PVR), retinal arterial or venous occlusive disease,retinal detachment, uveitic retinal disease; sympathetic ophthalmia;Vogt-Koyanagi-Harada (VKH) syndrome; uveal diffusion; a posterior ocularcondition caused by or influenced by an ocular laser treatment;posterior ocular conditions caused by or influenced by a photodynamictherapy, photocoagulation, radiation retinopathy, epiretinal membranedisorders, branch retinal vein occlusion, anterior ischemic opticneuropathy, non-retinopathy diabetic retinal dysfunction, retinitispigmentosa, and glaucoma. Glaucoma can be considered a posterior ocularcondition because the therapeutic goal is to prevent the loss of orreduce the occurrence of loss of vision due to damage to or loss ofretinal ganglion cells or retinal nerve fibers (i.e., neuroprotection).

In some embodiments, the ophthalmic disorder is ocular inflammationresulting from, e.g., iritis, conjunctivitis, seasonal allergicconjunctivitis, acute and chronic endophthalmitis, anterior uveitis,uveitis associated with systemic diseases, posterior segment uveitis,chorioretinitis, pars planitis, masquerade syndromes including ocularlymphoma, pemphigoid, scleritis, keratitis, severe ocular allergy,corneal abrasion and blood-aqueous barrier disruption. In yet anotherembodiment, the ophthalmic disorder is post-operative ocularinflammation resulting from, for example, photorefractive keratectomy,cataract removal surgery, intraocular lens implantation, vitrectomy,corneal transplantation, forms of lamellar keratectomy (DSEK, etc), andradial keratotomy.

E. MESENCHYMAL STEM CELLS

Certain embodiments of the present invention involve administering to asubject with an eye disease a therapeutically effective amount of acomposition that includes a MSC. The MSC in the composition can bemodified or unmodified (i.e., the MSC can be biochemically and/orgenetically manipulated in any number of ways that are further describedherein, or can be used without biochemical manipulation (i.e.,stimulation) and/or without genetic modification. In one embodiment, theMSC in a therapeutic composition of the invention has been modulated tooverexpress a therapeutic protein as set forth herein.

Notwithstanding the foregoing description of targeted diseases andconditions for which the present invention can provide a prophylactic ortherapeutic benefit, it is considered that modified or unmodified MSCs,or combinations thereof, are useful for treatment of diseases whichinclude but are not necessarily limited to corneal disease, glaucoma andretinal disease. In particular embodiments, the modified or unmodifiedmesenchymal stem cells can be used for therapy of any stage or severityof glaucoma, or for corneal epithelial injury, or for corneal trauma, orfor age-related macular degeneration, or for retinitis pigmentosa, orfor Stevens-Johnson Syndrome, or for ocular cicatricial pemphigoid, orany combination thereof. In connection with age-related maculardegeneration, a preferred embodiment includes intravitreal and/orsubretinal injection of the mesenchymal stem cells.

1. MSCs Generally

Based upon the disclosure provided herein, MSCs can be obtained from anysource. The MSCs may be autologous with respect to the recipient(obtained from the same host) or allogeneic with respect to therecipient. In addition, the MSCs may be xenogeneic to the recipient(obtained from an animal of a different species), for example rat MSCsmay be used to suppress inflammation in a human.

In a further non-limiting embodiment, MSCs used in the present inventioncan be isolated, from the bone marrow of any species of mammal,including but not limited to, human, mouse, rat, ape, gibbon, bovine. Ina non-limiting embodiment, the MSCs are isolated from a human, a mouse,or a rat. In another non-limiting embodiment, the MSCs are isolated froma human.

Based upon the present disclosure, MSCs can be isolated and expanded inculture in vitro to obtain sufficient numbers of cells for use in themethods described herein provided that the MSCs are cultured in a mannerthat promotes aggregation and formation of spheroids. For example, MSCscan be isolated from human bone marrow and cultured in complete medium(DMEM low glucose containing 4 mM L-glutamine, 10% FBS, and 1%penicillin/streptomycin) in hanging drops or on non-adherent dishes.However, the invention should in no way be construed to be limited toany one method of isolating and culturing medium. Rather, any method ofisolating and culturing medium should be construed to be included in thepresent invention provided that the MSCs are cultured in a manner thatpromotes aggregation and formation of spheroids.

Any medium capable of supporting MSCs in vitro may be used to culturethe MSCs. Media formulations that can support the growth of MSCsinclude, but are not limited to, Dulbecco's Modified Eagle's Medium(DMEM), alpha modified Minimal Essential Medium (αMEM), and Roswell ParkMemorial Institute Media 1640 (RPMI Media 1640) and the like. Typically,0 to 20% fetal bovine serum (FBS) or 1-20% horse serum is added to theabove medium in order to support the growth of MSCs. A defined medium,however, also can be used if the growth factors, cytokines, and hormonesnecessary for culturing MSCs are provided at appropriate concentrationsin the medium. Media useful in the methods of the invention may containone or more compounds of interest, including but not limited toantibiotics, mitogenic or differentiation compounds useful for theculturing of MSCs. The cells may be grown in one non-limitingembodiment, at temperatures between 27° C. to 40° C., in anothernon-limiting embodiment at 31° C. to 37° C., and in another non-limitingembodiment in a humidified incubator. The carbon dioxide content may bemaintained between 2% to 10% and the oxygen content may be maintainedbetween 1% and 22%; however, the invention should in no way be construedto be limited to any one method of isolating and culturing MSCs. Rather,any method of isolating and culturing MSCs should be construed to beincluded in the present invention.

Antibiotics which can be added into the medium include, but are notlimited to, penicillin and streptomycin. The concentration of penicillinin the culture medium is about 10 to about 200 units per ml. Theconcentration of streptomycin in the culture medium is about 10 to about200 μg/ml.

2. Mesenchymal Stem Cells Modified to Express a Therapeutic Protein

In a non-limiting embodiment, the mesenchymal stem cells are culturedunder conditions which, as noted hereinabove, provide for theaggregation of the mesenchymal stem cells into a spheroidal aggregate,and provide for optimal expression of the therapeutic protein(s).

In one non-limiting embodiment, the mesenchymal stem cells are culturedin a medium, such as complete culture medium (CCM), for example, whichincludes serum in an amount effective to upregulate one or more of thehereinabove noted therapeutic proteins. For example, the medium mayinclude fetal bovine serum in an amount of up to 20%. In a non-limitingembodiment, the fetal bovine serum is present in an amount of about 17%.The mesenchymal stem cells are cultured under conditions and for aperiod of time (for example, 7 or 8 days) sufficient to provide asufficient number of cells for further culturing. The culture medium mayinclude growth factors other than or in addition to serum to upregulateone or more of the hereinabove noted therapeutic proteins.

In one non-limiting embodiment, the spheroids can be prepared byculturing the MSCs on bacterial plates (as distinct from plates treatedfor culture of animal cells) so that the MSCs aggregate spontaneouslyinto spheroids (Bartosh, 2010).

The cells then are cultured under conditions which promote the formationof spheroidal aggregates of the cells. In one non-limiting embodiment,the cells are cultured as hanging drops. Each drop of cells containsmesenchymal stem cells in an amount which provides for optimalexpression of the at least one therapeutic protein. In a non-limitingembodiment, the hanging drops of the cells are cultured in a medium,such as complete culture medium, containing fetal bovine serum in anamount of up to 20%. In a non-limiting embodiment, the fetal bovineserum is present in an amount of about 17%.

In another non-limiting embodiment, each hanging drop of mesenchymalstem cells that is cultured contains from about 10,000 to about 500,000cells/drop. In another non-limiting embodiment, each hanging drop ofmesenchymal stem cells that is cultured contains from about 10,000 toabout 250,000 cells/drop. In a further non-limiting embodiment, eachhanging drop of cells contains from about 10,000 to about 25,000cells/drop. In yet another non-limiting embodiment, each hanging drop ofcells contains about 25,000/drop.

The hanging drops of mesenchymal stem cells are cultured for a period oftime sufficient for forming spheroidal aggregates of the mesenchymalstem cells. In general, the drops of cells are cultured for a period oftime of up to 4 days.

Once the spheroidal aggregates of the mesenchymal stem cells are formed,the mesenchymal stem cells may, if desired, be dissociated from thespheroids by incubating the spheroids in the presence of a dissociationagent, such as trypsin and/or EDTA, for example.

The invention comprises the treatment of an MSC in culture to expresstherapeutic proteins that are effective in treating a disease of theeye. For example, the MSCs can be cultured in the presence of TNF-α. Insome instances, the MSCs can be pre-activated by culturing in thepresence of IFN-μγ. In other instances, the MSCs can be pre-activated byculturing in the presence of IL-1B. In some instances, MSCs can bepre-activated using any combination of TNF-α, IFN-μ, and IL-1B.Pre-activation of the MSCs induce the cells to secrete therapeuticproteins. Thus, the MSCs themselves, the secreted proteins, or thecombination of both provide a source of a therapeutic composition. Inone embodiment, a recombinant version of the therapeutic proteinsecreted from the pre-activated MSCs can be used as a therapeuticcomposition.

In some instances, the MSCs are contacted with an agent that inducesMSCs to secrete therapeutic proteins in a culturing medium. Theculturing medium generally comprises a base media. Non-limiting examplesof base media useful in the methods of the invention include MinimumEssential Medium Eagle, ADC-1, LPM (Bovine Serum Albumin-free), F10(HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and withoutFitton-Jackson Modification), Basal Medium Eagle (BME—with the additionof Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM-withoutserum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM),Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199 (M199E—withEarle's salt base), Medium M199 (M199H—with Hank's salt base), MinimumEssential Medium Eagle (MEM-E—with Earle's salt base), Minimum EssentialMedium Eagle (MEM-H—with Hank's salt base) and Minimum Essential MediumEagle (MEM-NAA with non essential amino acids), among numerous others,including medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB75261, MAB 8713. DM 145, Williams' G, Neuman & Tytell, Higuchi, MCDB301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153. A preferredmedium for use in the present invention is DMEM. These and other usefulmedia are available from GIBCO, Grand Island, N.Y., USA and BiologicalIndustries, Bet HaEmek, Israel, among others. A number of these mediaare summarized in Methods in Enzymology, Volume LVIII, “Cell Culture”,pp. 62-72, edited by William B. Jakoby and Ira H. Pastan, published byAcademic Press, Inc.

Additional non-limiting examples of media useful in the methods of theinvention can contain fetal serum of bovine or other species at aconcentration of at least 1% to about 30%, preferably at least about 5%to 15%, mostly preferably about 10%. Embryonic extract of chicken orother species can be present at a concentration of about 1% to 30%,preferably at least about 5% to 15%, most preferably about 10%.

In a non-limiting embodiment, the MSCs are isolated from the mammal intowhich the treated MSC are to be introduced; however, the MSCs may alsobe isolated from an organism of the same or different species as themammal

F. GENETIC MODIFICATION OF THERAPEUTIC CELLS

In some embodiments of the present invention the subject is administereda therapeutically effective amount of cells that have been modified tooverexpress a therapeutic protein of the present invention. For example,the cells of the invention may be transformed stably or transiently witha nucleic acid of interest prior to introduction into the eye of themammal Nucleic acid sequences of interest include, but are not limitedto those encoding gene products TSG-6 and biologically active fragmentsand analogs thereof, and STC-1 and biologically active fragments andanalogs thereof. Methods of transformation of cells such as MSCs areknown to those skilled in the art, as are methods for introducing cellsinto a bone at the site of surgery or fracture.

In cases in which a gene construct is transfected into a cell, theheterologous gene is linked operably to regulatory sequences required toachieve expression of the gene in the cell. Such regulatory sequencestypically include a promoter and a polyadenylation signal. In particularembodiments, the gene expresses a protein as set forth in any of Tables1-3 or an amino acid sequence that has at least 95% sequence identity toa protein as set forth in any of Tables 1-3.

In a non-limiting embodiment, the gene construct is provided as anexpression vector that includes the coding sequence for a heterologousprotein operably linked to essential regulatory sequences such that whenthe vector is transfected into the cell, the coding sequence will beexpressed by the cell. The coding sequence is linked operably to theregulatory elements necessary for expression of that sequence in thecells. The nucleotide sequence that encodes the protein may be cDNA,genomic DNA, synthesized DNA or a hybrid thereof, or an RNA moleculesuch as mRNA.

The gene construct includes the nucleotide sequence encoding thebeneficial protein is linked operably to the regulatory elements and mayremain present in the cell as a functioning cytoplasmic molecule, afunctioning episomal molecule, or it may integrate into the cell'schromosomal DNA. Exogenous genetic material may be introduced into cellswhere it remains as separate genetic material in the form of a plasmid.Alternatively, linear DNA which can integrate into the chromosome may beintroduced into the cell. When introducing DNA into the cell, reagentswhich promote DNA integration into chromosomes may be added. DNAsequences which are useful to promote integration may also be includedin the DNA molecule. Alternatively, RNA may be introduced into the cell.

The cells of the present invention can be transfected using well knowntechniques readily available to those having ordinary skill in the art.Exogenous genes may be introduced into the cells using standard methodswhere the cell expresses the protein encoded by the gene. In someembodiments, cells are transfected by calcium phosphate precipitationtransfection, DEAE dextran transfection, electroporation,microinjection, liposome-mediated transfer, chemical-mediated transfer,ligand mediated transfer or recombinant viral vector transfer.

In some embodiments, vectors are used to introduce DNA with desiredsequences into the cell. The vector may be any vector as set forth inthe foregoing sections.

The MSCs, in a non-limiting embodiment, may have one or more genesmodified or may be treated such that the modification has the ability tocause the MSCs to self-destruct or “commit suicide” because of suchmodification, or upon presentation of a second drug (e.g., a prodrug) orsignaling compound to initiate such destruction of the MSCs.

G. THERAPY

1. General

Some embodiments of the present invention include methods of using cellsexpressing a polypeptide as set forth herein as a therapy to inhibitinflammation in the context of an ocular disease.

Cells can be suspended in an appropriate diluent. Suitable excipientsfor injection solutions are those that are biologically andphysiologically compatible with the cells and with the recipient, suchas buffered saline solution or other suitable excipients. Thecomposition for administration can be formulated, produced and storedaccording to standard methods complying with proper sterility andstability.

The dosage of the cells varies within wide limits and may be adjusted tothe individual requirements in each particular case. The number of cellsused depends on the weight and condition of the recipient, the numberand/or frequency of administrations, and other variables known to thoseof skill in the art, including, but not limited to, the age and sex ofthe patient, the disease or disorder being treated, and the extent andseverity thereof.

In particular embodiments, the therapeutic polypeptide is a humantherapeutic polypeptide. In other embodiments, the polypeptide isderived from a non-human source, such as a mouse, rat, rabbit, horse,cow, or primate.

For treatment of eye disease, the therapeutic cells, polypeptides, orpolynucleotides may be administered using any method known to those ofordinary skill in the art. For example, administration may beintravenous, intracameral, intravitreous, subconjunctival, sub-Tenon's,subretinal, or topical to the corneal surface. Administration may beintraoperative in some embodiments, such as by injection into the eye.The therapeutic polypeptides of the present invention may be delivereddirectly to the eye (for example: topical ocular drops or ointments;slow release devices in the cul-de-sac or implanted adjacent to thesclera or within the eye; periocular, conjunctival, sub-tenons,intracameral, intravitreal, or intracanalicular injections) orsystemically (for example: orally, intravenous, subcutaneous orintramuscular injections; parenterally, dermal or nasal delivery) usingtechniques well known by those skilled in the art. It is furthercontemplated that the agents of the invention may be formulated inintraocular insert or implant devices.

Often, treatment dosages are titrated upward from a low level tooptimize safety and efficacy. Generally, daily dosages will fall withina range of about 0.01 to 20 mg protein per kilogram of body weight.Typically, the dosage range will be from about 0.1 to 5 mg protein perkilogram of body weight. In some embodiments for intravitreal injection,a dose includes 0.01 to 5 mg of polypeptide. In more specificembodiments, a dose includes between 0.1 and 2.0 mg. The polypeptide isformulated in a carrier volume ranging from 0.2 ml to 5 ml. In moreparticular embodiments, the carrier volume is between 0.5 and 2.0 ml.Nonlimiting examples of carriers include sterile water, normal saline,phosphate buffered saline, and optionally including any of theexcipients set forth herein.

Various modifications or derivatives of the proteins, such as additionof polyethylene glycol chains (PEGylation), may be made to influencetheir pharmacokinetic and/or pharmacodynamic properties.

To administer the protein by other than parenteral administration, theprotein may be coated or co-administered with a material to prevent itsinactivation. For example, the protein may be administered in anincomplete adjuvant, co-administered with enzyme inhibitors oradministered in liposomes. Enzyme inhibitors include pancreatic trypsininhibitor, disopropylfluorophosphate (DEP) and trasylol. Liposomesinclude water-in-oil-in-water, CGF emulsions, as well as conventionalliposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

The compositions of the present invention optionally comprise one ormore excipients. Excipients commonly used in solution compositionsintended for topical application to the eyes, such as solutions,include, but are not limited to, tonicity agents, preservatives,chelating agents, buffering agents, surfactants and antioxidants.Suitable tonicity-adjusting agents include mannitol, sodium chloride,glycerin, sorbitol and the like. Suitable preservatives includep-hydroxybenzoic acid ester, benzalkonium chloride, benzododeciniumbromide, polyquaternium-1 and the like. Suitable chelating agentsinclude sodium edetate and the like. Suitable buffering agents includephosphates, borates, citrates, acetates, tromethamine, and the like.Suitable surfactants include ionic and nonionic surfactants, thoughnonionic surfactants are preferred, such as polysorbates,polyethoxylated castor oil derivatives, polyethoxylated fatty acids,polyethoxylated alcohols, polyoxyethylene-polyoxypropylene blockcopolymers, and oxyethylated tertiary octylphenol formaldehyde polymer(tyloxapol). Suitable antioxidants include sulfites, thiosulfate,ascorbates, BHA, BHT, tocopherols, and the like. The compositions of thepresent invention optionally comprise an additional active agent. Thecompositions of the present invention may contain one or more nonionic,anionic, or cationic polymers as lubricants or as viscosity agents,including but not limited to hydroxypropyl methylcelluloses (HPMCs),methylcelluloses, carboxymethylcelluloses (CMCs), polyethylene glycols(PEGs), poloxamers, polypropylene glycols, xanthan gums, guar gums,carbomers, polyvinyl alcohols (PVAs), polyvinylpyrrolidones (PVPs),alginic acids and salts, gellan gums, carrageenans, and chitosans.

An “effective amount” of a therapeutic polypeptide, polynucleotide, orcells is an amount that will ameliorate one or more of the well knownparameters that characterize medical conditions of the eye disease to betreated.

Although the compositions of this invention can be administered insimple solution, they are more typically used in combination with othermaterials such as carriers, preferably pharmaceutical carriers. Usefulpharmaceutical carriers can be any compatible, non-toxic substancesuitable for delivering the compositions of the invention to a patient.Sterile water, alcohol, fats, waxes, and inert solids may be included ina carrier. Pharmaceutically acceptable adjuvants (buffering agents,dispersing agents) may also be incorporated into the pharmaceuticalcomposition. Generally, compositions useful for parenteraladministration of such drugs are well known; e.g., Remington'sPharmaceutical Science, 17th Ed. (Mack Publishing Company, Easton, Pa.,1990). Alternatively, compositions of the invention may be introducedinto a patient's body by implantable drug delivery systems [Urquhart etal., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984).

Therapeutic formulations may be administered in many conventional dosageformulations. Formulations typically comprise at least one activeingredient, together with one or more pharmaceutically acceptablecarriers. In some embodiments, the ophthalmic compositions areformulated to provide for an intraocular concentration of about 0.1-100nanomolar (nM) or, in a further embodiment, 1-10 nM. Peak plasmaconcentrations of up to 20 micromolar may be achieved for systemicadministration. Topical compositions are delivered to the surface of theeye one to four times per day according to the routine discretion of askilled clinician. The pH of the formulation should be 4-9, or 4.5 to7.4. Systemic formulations may contain about 10 mg to 1000 mg, about 10mg to 500 mg, about 10 mg to 100 mg or to 125 mg, for example, of thetherapeutic protein.

The formulations conveniently may be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. See,e.g., Gilman et al. (eds.) (1990), The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, supra, Easton, Pa.; Avis, et al. (eds.) (1993) PharmaceuticalDosage Forms: Parenteral Medications Dekker, N.Y.; Lieberman et al.(eds.) (1990), Pharmaceutical Dosage Forms: Tablets, Dekker, N.Y.; andLieberman et al. (eds.) (1990), Pharmaceutical Dosage Forms: DisperseSystems, Dekker, N.Y.

In additional non-limiting embodiments, the present inventioncontemplates administration of the proteins by gene therapy methods,e.g., administration of an isolated nucleic acid encoding a protein ofinterest. The proteins of the present invention have beenwell-characterized, both as to the nucleic acid sequences encoding theproteins and the resultant amino acid sequences of the proteins.Engineering of such isolated nucleic acids by recombinant DNA methods iswell within the ability of one skilled in the art. Codon optimization,for purposes of maximizing recombinant protein yields in particular cellbackgrounds, also is well within the ability of one skilled in the art.

There are two major approaches for introducing a nucleic acid encodingthe protein (optionally contained in a vector) into a patient's cells;in vivo and ex vivo. For in vivo delivery, the nucleic acid is injecteddirectly into the patient, usually at the site where the protein isrequired. For ex vivo treatment, the patient's cells are removed, thenucleic acid is introduced into these isolated cells and the modifiedcells are administered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are avariety of techniques available for introducing nucleic acids intoviable cells. The techniques vary depending upon whether the nucleicacid is transferred into cultured cells in vitro, or in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. Commonly used vectors for ex vivodelivery of the gene are retroviral and lentiviral vectors.

Preferred in vivo nucleic acid transfer techniques include transfectionwith viral vectors such as adenovirus, Herpes Simplex I virus,adeno-associated virus, lipid-based systems (useful lipids forlipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample), naked DNA, and transposon-based expression systems. For reviewof the currently known gene marking and gene therapy protocols seeAnderson, et al., Science 256:808-8 13 (1992). See also WO 93/25673 andthe references cited therein.

Therapeutic compositions and formulations thereof of the invention canbe used, for example, for reducing inflammation due to seasonal orbacterial conjunctivitis, for reducing post-surgical pain andinflammation, to prevent or treat inflammatory tissue damage associatedwith fungal or bacterial infections of the eye, to treat herpes zosterophthalmicus, to reduce intraocular pressure, or to treatendophthalmitis.

More particularly, in one non-limiting embodiment, the present inventionprovides a method for treating an ophthalmic disorder in a mammal (e.g.,including human and non-human primates), the method comprisingadministering to the eye of the mammal a therapeutically effect amountof a formulation of the present invention comprising a lipid phase, anaqueous phase and a therapeutic agent as hereinabove described, whereinthe therapeutic agent is useful for treating the ophthalmic disorder. Inone embodiment, the ophthalmic disorder is post-operative pain.

In employing the liposome formulations of the present invention, in anon-limiting embodiment, administration is ocularly, which term is usedto mean delivery of therapeutic agents through the surface of the eye,including the sclera, the cornea, the conjunctiva and the limbus, orinto the anterior chamber or vitreous chamber of the eye. Oculardelivery can be accomplished by numerous means, for example, by topicalapplication of a formulation such as an eye drop, by injection, or bymeans of an electrotransport drug delivery system.

In another non-limiting embodiment, the therapeutic cells or therapeuticproteins employed for treating a disease or disorder of the eye may becontained in a nanoparticle. Such nanoparticles may be formed by methodsknown to those skilled in the art.

Such nanoparticles may be administered ocularly, i.e., through thesurface of the eye, including the sclera, cornea, conjunctiva, and thelimbus, or into the anterior chamber of the eye. Such ocularadministration may be accomplished by any of a variety of means,including, in a non-limiting embodiment, by topical application of aformulation such as an eye drop, by injection, or by means of anelectrotransport drug delivery system.

2. Combination Therapy

The compositions and/or cells of the present invention are suitable foruse in combination with other therapeutic agents for treatment of eyedisease. They can be comprised in the same formulation or in differentformulations. Non-limiting examples of other agents for use in thetreatment of eye disease contemplated by the present invention includeartificial tears, anti-glaucoma agents, such as beta-blockers includingtimolol, betaxolol, levobetaxolol, carteolol, miotics includingpilocarpine, carbonic anhydrase inhibitors, prostaglandins,serotonergics, muscarinics, dopaminergic agonists, adrenergic agonistsincluding apraclonidine and brimonidine; anti-angiogenesis agents;anti-infective agents including quinolones such as ciprofloxacin, andaminoglycosides such as tobramycin and gentamicin; non-steroidal andsteroidal anti-inflammatory agents, such as suprofen, diclofenac,ketorolac, rimexolone and tetrahydrocortisol; growth factors, such asEGF; immunosuppressant agents; and anti-allergic agents includingolopatadine; prostaglandins such as latanoprost; 15-keto latanoprost;travoprost; and unoprostone isopropyl.

H. EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations which are evident as a result of the teachings providedherein. Those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 MSCs and MSC-Derived Factors Suppressed Inflammation andNeovascularization, and Promoted Wound Healing in Chemically-Injured RatCornea

Beneficial effects of MSCs and MSC-derived factors have been observed insuppressing corneal inflammation/neovascularization and promoting woundhealing (Oh et al., 2008). Corneal inflammation, neovascularization, anddelayed wound healing were induced in rats by applying 100% ethanol for30 sec and scraping both the epithelium in the limbus and the wholecornea. The reliability and reproducibility of this model was previouslyconfirmed and repetitively used by other researchers (Cho et al., 1998;Avila et al., 2001; Ti et al., 2002; Espana et al., 2003; Homma et al,2004; Oh et al., 2009b). Massive infiltration of inflammatory cells andgrowth of new vessels in cornea were induced in this model (FIG. 1).

Immediately after injury, rat MSCs or conditioned media (CM) derivedfrom MSC cultures were put into an applicator and allowed to remain inthe damaged cornea for two hours. A 6-mm-diameter hollow tube was usedas an applicator (FIG. 2). This method of application has previouslybeen proven effective for applying stem cells to cornea (Homma et al.,2004; Ueno et al., 2007). The following were the four groups that werestudied and subjected to different treatments: (1) 200 μL of fresh media(control group, n=10), (2) 200 μL of supernatants collected from theMSCs culture (MSC-CM I group, n=10), (3) 200 μL MSC-CM applied for twohours a day over three consecutive days (MSC-CM II group, n=10), (4) 200μL of media containing 2×10⁶MSCs (MSC group, n=10).

Effects of MSCs or MSC-CM on the cornea were determined in three ways:(I) gross examination by slit-lamp biomicroscopy based on the findingsof transparency, neovascularization (NV), and epithelial defects, (2)histological analysis for infiltration of inflammatory cells(hematoxylin-eosin staining) or CD4+ T cells (immunofluorescentstaining), and (3) ELISA and real time PCR assays for inflammation- andangiogenesis-related cytokines. As a result, it was found that cornealinflammation and NV rapidly decreased in MSC-treated corneas over timeafter injury, while they gradually increased in the vehicle-treatedcontrols (FIG. 3). The degree of corneal inflammation and NV was thelowest in the MSC group and the highest in the control group. Notably,it was observed that MSC-CM was also effective in reducing cornealinflammation and NV in proportion to the number of MSC-CM applications.More specifically, corneas treated three times with MSC-CM (MSC-CM IIgroup) were more transparent compared to both the controls and thecorneas that were treated only once with MSC-CM (MSC-CM I group).Re-epithelialization was faster in the corneas treated with MSCs andMSC-CM.

Similar to clinical findings, histological analysis (FIG. 4) revealedthat MSCs and MSC-CM II groups had fewer inflammatory cell infiltratesthan the control and MSC-CM I groups. Comparisons between MSCs andMSC-CM II groups showed that corneas treated with MSCs had significantlyfewer inflammatory cells than those treated with MSC-CM three times.

ELISA showed that production of proinflammatory cytokines IL-2 and IFN-μwas decreased in MSC- and MSC-CM-treated corneas (FIG. 5 A, B). Incontrast, large quantities of anti-inflammatory cytokines IL-10 andTGF-B were detected in MSC-treated corneas (FIG. 5 C, D)

In order to determine the mechanisms associated with regression of newvessels by MSCs, the expression of angiogenesis-related cytokines,TSP-1, MMP-2, MMP-9, and VEGF was evaluated. Real time RT-PCR revealedthat the level of an anti-angiogenic factor, TSP-1, was significantlyupregulated in MSC and MSC-CM II groups (FIG. 6). The expression ofpro-angiogenic factors, MMP-2 and MMP-9, was significantly repressed inMSC-treated corneas, compared to control corneas.

Example 2 Human MSC-Conditioned Media Rescued Human Corneal EpithelialCells from Chemically-Induced Apoptosis

Human corneal epithelial cells (hCECs) were chemically damaged byincubation in 15% ethanol for 30 seconds. Damaged hCECs were culturedwith one of the following: (1) hMSC-conditioned media, (2) conditionedmedia from hMSC-damaged hCECs coculture, or (3) fresh media. Then,survival of hCECs was evaluated with MTT assay. The result showed thatthe proportion of damaged hCECs was significantly decreased whencultured with hMSC-conditioned media (FIG. 7).

Example 3 Production of MMP-9 is Significantly Suppressed inChemically-Damaged Human Corneal Epithelial Cells by hMSCs

The following experiments were performed to evaluate how MSCs affectedcorneal epithelial cells in terms of inflammatory and angiogeniccytokine secretion. The hCECs were chemically damaged, then they werecocultured with hMSCs for 24 hours, and finally the cell-freesupernatant was analyzed for cytokine concentration by ELISA. Thecoculture groups were as follows: (I) hPBMCs (human peripheral bloodmononuclear cells), (2) hCECs, (3) hPBMCs/hCECs, (4) hMSCs, (5)hMSCs/hPBMCs, (6) hMSCs/hCECs, and (7) hMSCs/hPBMCs/hCECs. As a result,it was observed that MSCs constitutively secreted VEGF, MMP-2, and TSP-1(FIG. 8). It is important to note that MMP-9, which is highly secretedby damaged hCECs, was significantly suppressed by hMSCs (FIG. 8, upperright). In fact, as a consequence of hMSC suppression the level of MMP-9was reduced from 100% to 8%. Based on these results, it was believedthat the suppression of MMP-9, a key player in corneal inflammation,angiogenesis, and wound healing would be one of the mechanismsresponsible for MSC action during corneal regeneration. MMP-9 is one ofthe pro-inflammatory proteases that has its activation significantlyinhibited by TSG-6 (Milner and Day, 2003; Milner et al., 2006).

The results presented herein demonstrate that application of either MSCsor conditioned medium from the MSCs decreased inflammation andneovascularization in a rat model for noninfectious inflammation of thecornea. Without wishing to be bound by any particular theory, thebeneficial effects of the MSCs are at least in part explained by theeffects of decreasing the levels of inflammatory cytokines andinflammation-related proteases both in the rat model of corneal damageand in cocultures with conical epithelial cells. Therefore the data areconsistent with the hypothesis that the beneficial effects of MSCs areexplained by the production by the cells of the anti-inflammatoryprotein TSG-6 and/or the anti-apoptotic protein STC-1.

Example 4 Use of Pre-Activated MSCs for the Treatment of Cornea

The following experiments were designed to test whether MSCspreactivated in culture to express therapeutic proteins would be moreeffective in reducing inflammation and neovascularization followingchemically-induced injury to the cornea than standard cultures of MSCs.The experiments were set up to compare standard preparations of MSCswith MSCs pre-activated in culture with TNFα (FIGS. 10 C and D) in therat model in terms of the minimum number of cells required to produce(a) significant improvements in neovascularization, opacity, epithelialdefects, and infiltration of inflammatory cells as in FIGS. 3 and 4; (b)significant decreases in the inflammatory cytokine IFN-μ (FIG. 5); and(c) significant decreases in the inflammation-related proteases MMP-2and MMP-9 (FIG. 6). In parallel standard preparations and pre-activatedMSCs by intracameral injection (IC; into anterior chamber) in the modelare compared using the same measures of effectiveness.

As summarized in Table 4, the initial experiments are carried out withboth rat MSCs and human MSCs (hMSCs), since the hMSCs are more relevantto the potential clinical applications of the results, and human androdent MSCs have proven to be equally effective in other rodent models(Lee et al., 2009; Block et al.; 2009; Ohtaki et al., 2008; Iso et al.,2007; Ortiz et al., 2007; Lee et al., 2006; Munoz et al., 2006).

TABLE 4 MSCs Delivery Dose # Rats 1. Species hMSCs Topical 2 × 10⁴ 10 2× 10⁵ 10 2 × 10⁶ 10 rMSCs Topical 2 × 10⁴ 10 2 × 10⁵ 10 2 × 10⁶ 10 2.Route hMSCs IC 2 × 10³ 10 2 × 10⁴ 10 2 × 10⁵ 10 rMSCs IC 2 × 10³ 10 2 ×10⁴ 10 2 × 10⁵ 10 3. acMSCs Ac h/rMSCs Topical 2 × 10⁴ 10 2 × 10⁵ 10 2 ×10⁶ 10 Ac h/rMSCs IC 2 × 10³ 10 2 × 10⁴ 10 2 × 10⁵ 10 Total 180The materials and methods employed in the experiments disclosed hereinare now described.Corneal Surface Inflammation Model

Corneal surface inflammation can be created in rats by application of100% ethanol and mechanical debridement of corneal and limbal epitheliumusing the protocol described in Oh et al. (2008). This model induces theinfiltration of neutrophils, macrophages, neovascularization, anddelayed wound healing in cornea (FIG. 1).

MSC's

Human MSCs (hMSCS) and rat MSCs (rMSCs) can be acquired fromstandardized preparations currently being distributed by our NIH/NCRRfunded center (P40 RR 17447). For hMSCs, the standardized cells can befurther screened to select preparations that are over 90% positive forPODXL. PODXL serves as a marker for MSCs that are likely to expressother epitopes (c-MET, CXCR4, and CXC3CR1) for early progenitors invivo. For rMSCs, MSCs can be isolated from the bone marrow of Lewis rats(Javazon et al., 2001).

Route of Treatment Delivery

Topical application (FIG. 2) and IC injection are compared. Thiscomparison allows for the determination of whether IC injection iseffective at lower doses than topical application. For topical delivery,a hollow tube can be applied to the cornea, the cells (200 μL) can beput into the tube, and allowed to remain in the cornea for two hours(FIG. 2). The dose of cells can be varied from 2×10⁴ to 2×10⁶, the dosepreviously found to be effective (FIGS. 3 to 6). Topical application canbe repeated on consecutive days for a total of three treatments. For ICinjection, the media (5 μL) can be injected once into the anteriorchamber of the rat eye. The dose of cells can be varied from 2×10³ to2×10⁵, i.e., to the maximal concentration that can be employed withoutaggregation of the cells (Lee et al., 2009).

Assays for Corneal Surface Regeneration

The eyes are examined with slit-lamp biomicroscopy and recorded withphotography once a week. The clinical outcome is graded by a blindedinvestigator who is an ophthalmologist under the following criteria: (1)corneal epithelial integrity, (2) transparency, and (3)neovascularization (NV). A 1% fluorescein sodium solution can be used toevaluate the degree of corneal epithelial defects. Subsequently, defectsare quantified by the ratio of epithelial defect area to total cornealarea, using an image analyzer. The corneal clarity is graded from 0 to 4using the method in Fantes et al. (1990) and Oh et al. (2008). Thecorneal NV are quantified by calculating the area of vessel growth withthe method used in D'Amato et al. (1994), Oh et al. (2008), and Oh etal. (2009b). After three weeks, corneas are excised, and examined usingappropriate assays discussed elsewhere herein.

Assays for Corneal Inflammation

Corneas are either stained with hematoxylin-eosin or subjected toimmunostaining with neutrophil- and macrophage-specific markers. Thenumbers of inflammatory cells are counted on the H&E-stained slides, Thenumbers of positively-stained cells are counted on the immunostainedslides. Also, the infiltration and accumulation of neutrophils in thecornea are quantitated by measuring myeloperoxidase (MPO) activity withthe MPO sandwich ELISA assay (Armstrong et al., 1998).

Assays for Modulation of Inflammation-, Angiogenesis-, andApoptosis-Related Molecules

The expression of proteins for inflammation-related factors (IL-1B,IL-2, IFN-μ, IL-6, IL-10, TGF-B1), angiogenesis-related factors (TSP-1,VEGF), apoptosis-related factors (Fas/Fas ligand), and gelatinases(MMP-2, MMP-9) are measured in corneas using ELISA assay.

The results of these experiments are now described. Without wishing tobe bound by any particular theory, it is believed that (1) hMSCs are aseffective in a rat model of corneal inflammation as rMSCs; (2) thepre-activated MSCs are more effective than the standard preparations ofMSCs; (3) one-time IC injection of MSCs are effective at lower dosesthan topical application.

It is possible that human proteins from hMSCs may not be as effective inthe rat model as rMSCs. However, it is believed that hMSCs are aseffective as rMSCs, because proteins are highly conserved acrossspecies, and it has been previously found that hMSCs worked in variousmurine models of inflammation (Lee et al., 2009; Block et al.; 2009;Ohtaki et al. 2008; Iso et al., 2007; Ortiz et al., 2007; Lee et al.,2006; Munoz et al., 2006). However, if it is observed that hMSCs areineffective in rats, rMSCs can be used instead of hMSCs.

MSCs activated with TNF-α may either be ineffective or may produce toxiceffects in cornea. It has been observed in preliminary experiments thatthe activation of hMSCs with TNF-α significantly upregulated somefactors to modulate inflammation and other factors to increase cellsurvival (Lee et al., 2009). However, if TNF-α activation is foundineffective, MSCs can be pre-activated with IFN-μ, IL-1B, or theircombinations as reported previously (Ren, et al., 2008). If ineffective,unmodified MSCs can be used for further experiments.

Example 5 Inflammation and Neovascularization of the Cornea can beReduced by Application of Two of the Therapeutic Proteins Produced byActivated MSCs: The Anti-Inflammatory Protein TSG-6 and theAnti-Apoptotic Protein STC-1

The following experiments were designed to assess the effectiveness ofusing TSG-6 and STC-1 for treating inflammation and neovascularizationof the cornea. Administration of recombinant proteins is an alternativeto administering MSCs to the mammal in need thereof. The experiments arecarried out as summarized in Table 5.

TABLE 5 Expt. Protein Delivery Dose # Rats 1. TSG-6 Topical 1 μg 10 10μg 10 100 μg 10 2. STC-1 Topical 1 μg 10 10 μg 10 100 μg 10 3. TSG-6 +STC-1 (1:1) Topical 1 μg 10 10 μg 10 100 μg 10 4. TSG-6 IC 0.1 μg 10 1μg 10 10 μg 10 5. STC-1 IC 0.1 μg 10 1 μg 10 6. TSG-6 + STC-1 (1:1) IC0.1 μg 10 1 μg 10 10 μg 10 Total 180

Briefly, recombinant TSG-6 (R & D Systems, Minneapolis, Minn.),recombinant STC-1 (BioVendor Laboratory Medicine, Inc.; Czech Republic),and their combinations are applied, respectively, to rat eyes using anappropriate delivery method. If topical application is used, the dosesof TSG-6 can be varied from 1 to 100 μg (Lee et al., 2009).Alternatively, if IC injection is used, dosage from 0.1 to 10 μg can beused. The same range of doses are also tested with recombinant STC-1.The maximally effective dose of each protein are determined. Then theproteins are prepared in 1:1 mixtures and the maximally effective doseagain are determined.

Without wishing to be bound by any particular theory (1) administrationof either TSG-6 or STC-1 is effective in suppressing cornealinflammation and promoting epithelial wound healing in adosage-dependent manner; (2) alternatively, administration of 1:1mixtures may be effective at lower doses because of synergistic effectsof the proteins; (3) intracameral administration of either or bothproteins may be effective in lower doses than topical application.

Example 6 Novel Therapeutic Factors Produced by hMSCs in Response toCorneal Injury

The invention is not limited to only TSG-6 and STC-1. That is, thecurrently available data do not exclude the hypothesis that MSCs producetheir beneficial effects on tissue repair by expression of othertherapeutic genes. Therefore, experiments can be designed to test foradditional therapeutic genes expressed by MSCs in the rat model forcorneal injury. As indicated in the accompanying scheme set forth inFIG. 14, the experiments can be carried out in three complementaryphases: Phase I: use hMSCs in the rat model for corneal injury, isolatethe total RNA from the treated corneas and assay the total RNA onspecies-specific microarrays followed by filtering the data fromcross-hybridization of the probes. Phase II: repeat the in vivoexperiments using rat MSCs that express GFP (available from our NCRR/NIHcenter for distribution of MSCs), enzymatically digest the samples ofcornea, isolate the GFP-expressing rMSCs by FACS sorting, and analyzethe RNA with a rat-specific microarray. Phase III: carry out co-cultureexperiments in transwells with injured corneal epithelial cells (FIGS. 7and 8) so that the RNA from the MSCs and the target cells can beisolated separately. The microarray data from the three phases of theexperiments can be used to identify candidate therapeutic genes usingthe following criteria: (i) genes upregulated by hMSCs or rMSCs byincubation with injured cornea or corneal epithelial cells, (ii) genesfor secretory proteins, and (iii) genes whose functions suggest thatthey may have anti-inflammatory or immunosuppressive effects. Verifyingthe roles of the candidate genes can be done using real-time RT-PCR,ELISAs or Western blots, knock down of the specific genes in MSCs withsiRNAs or lentiviruses, blocking antibodies, and replacement of the MSCsby the recombinant proteins (FIGS. 10 through 13).

The materials and methods employed in the experiments disclosed hereinare now described.

Assays for hMSCs in the Rat Model for Corneal Injury

Immediately after injury, the hMSCs (2×10⁶ cells/200 μI) are placed intoan applicator and allowed to remain on the damaged cornea for two hours(FIG. 2A). Eyelids are sutured for rats not to blink in order to preventthe shedding of transplanted cells. One day, one week, two weeks, andthree weeks later, the corneas are excised, homogenized (PowerGen;Fisher Scientific), extraction of DNA (Phase Lock Gel;Eppendor/Brinkmann Instruments) and the DNA assayed for the highlyrepetitive Alu sequences, unique to the human genome, to follow the fateof hMSCs in rat eyes (FIG. 9B). The use of Alu sequences provides ahighly sensitive assay since there are about 500,000 copies per cell.However, preliminary experiments indicated that use of conventionalprocedures greatly overestimated the content of human cells. Therefore,an improved protocol based on the chemical assay for extracted DNA andstandard curves for real-time RT-PCR of each tissue is used, The assayfor Alu sequences is complemented with a less sensitive assay extractedRNA (Trizol reagent, Invitrogen) human mRNA for GAPDH in order to assaylive human cells in the rat tissues (FIG. 9D). The results providequantitative data on the engraftment of the hMSCs.

Assays of Human and Rat mRNAs in Cornea by Microarrays

To examine the changes occurring in hMSCs applied in injured rat corneaand the changes in rat cornea caused by hMSCs, RNA will be isolated fromcornea (Trizol; Invitrogen), and assayed on both rat and humanmicroarrays (Affymetrix, Santa Clara, Calif.). Data will be filtered forcross-hybridization (Ohtaki et al., 2008; Lee et al., 2009), analyzedwith the dChip program, and normalized to a variance of 2 SD. Forcross-hybridization, three control groups were needed: (i) uninjured ratcornea treated with vehicle, (ii) injured rat cornea treated withvehicle, and (iii) uninjured rat cornea treated with hMSCs. After ananalysis of human genes upregulated 2 fold or more in hMSCs, candidategenes to confirm the data by human-specific real-time RT-PCR assays willbe selected.

Real-time RT-PCR and ELISA analysis

To determine whether the candidate genes are upregulated in culturedhMSCs, real-time RT-PCR is performed in cultured hMSCs. Double strandedcDNA is synthesized (SuperScript III; Invitrogen) and analyzed by realtime RT-PCR (ABI 7900, Sequence Detector; Applied Biosystems).Human-specific primers are obtained from commercial sources or designedfrom gene sequences. The data is further verified by human-specificELISA assays either from commercial sources or with kits developed fromcommercial antibodies. Alternatively, expression of the proteins areverified by Western blots where antibodies are available.

Confirmation with Recombinant Protein, siRNA, and Blocking Antibodies

Where recombinant protein is available for the candidate gene selectedabove, the protein is applied in a rat model to assess whether theprotein mimics the effects of MSCs. In addition, MSCs with a knock downof the selected gene either with an siRNA or lentivirus is tested (FIGS.11E and F). Also, where blocking antibodies are available, they can betested in the rat model (FIG. 13).

Assays from Co-Culture in Transwells

The experiments are performed with both hMSCs and rMSCs as described inFIGS. 7 and 8. The separate cell fractions are first assayed withmicroarrays and the roles of candidate genes confirmed as above.

The results of these experiments are now described. Without wishing tobe bound by any particular theory, it is believed that: (1) one or moreof the additional cornea-protective genes can be identified from themicroarray data; (2) the protein from the candidate gene(s) can beexpressed at increased levels in the rat model and the co-cultures withinjured lens epithelium; (3) the application of the candidate protein(s)is as effective in suppressing corneal inflammation as hMSCs; (4) bothhMSCs not expressing the gene and hMSCs combined with blocking antibodyfail to produce beneficial effects on rat model.

Example 7 The Anti-Inflammatory and Anti-Apoptotic Proteins from AdultStem/Progenitor Cells (MSCs) Protect the Cornea from Injury byPreventing Apoptosis and Suppressing Inflammation

The following experiments were designed to determine whetherinflammation of the cornea can be reduced by application of two of thetherapeutic proteins produced by activated MSCs: the anti-inflammatoryprotein TSG-6 and/or the anti-apoptotic protein STC-1.

To test the anti-apoptotic effect of STC-1 in vitro, human cornealepithelial progenitor cells (hCEPs) after exposure to ethanol werecultured for 24 hours with one of the following: (a) conditioned mediumfrom standard cultures of human MSCs (hMSCs), (b) conditioned mediumfrom hMSCs pre-activated to express therapeutic factors by incubationwith TNF-α for 24 hr, (c) rhSTC-1, (d) conditioned medium and blockingAb against rhSTC-1, or (e) IgG. Both fresh medium and the mediumconditioned from cultures of human dermal fibroblasts were used ascontrols. After incubation, the cell viability, proliferation andapoptosis were evaluated using MTT assay, BrdU uptake, and PI/annexinflow cytometry. It was observed that conditioned medium frompre-activated MSCs and rhSTC-1 had the greatest effects in hMSCsimproving the viability, increasing the proliferation, and inhibitingthe apoptosis of damaged hCEPs (FIG. 15).

To test the anti-inflammatory effect of TSG-6 in vivo, corneal surfaceinflammation was created in Lewis rats by ethanol application andmechanical debridement of corneal and limbal epithelium Immediatelyafter injury, rhTSG-6 (2 ng-2 ug) or the same volume of PBS was injectedinto the anterior chamber. Effects on the cornea were determined inthree ways: (1) gross examination by slit-lamp biomicroscopy based onthe findings of transparency and neovascularization (NV), (2)histological analysis for infiltration of inflammatory cell(hematoxylin-eosin staining), (3) myeloperoxidase (MPO) assay forinfiltration of neutrophils, (4) ELISA and real time PCR forinflammation-related chemokines and cytokines, (5) Gel zymography andELISA for total and active MMP-9. Changes in inflammatory markers insystemic circulation were also determined by WBC counting and serum MPOevaluation. It was observed that corneal opacity and neovascularizationwere significantly decreased in TSG-6-treated corneas compared toPBS-treated controls (FIG. 16). The infiltration of inflammatory cellsand expression of MMP-9 were significantly decreased in TSG-6-treatedcorneas compared to PBS-treated controls (FIG. 17). It was also observedthat TSG-6 up to the concentration of 0.002 ug/ml was effective inreducing corneal opacity, inflammation, and MMP-9 production (FIG. 19).Furthermore, it was observed that administration of TSG-6 decreased theexpression of inflammatory cytokines and chemokines (FIGS. 20, 21, 22).

The results presented herein demonstrate that therapeutic proteinsproduced by MSCs in response to an injury signal can protect the cornealsurface from damage by increasing the viability and proliferation ofcorneal epithelial progenitors and by suppressing inflammation incorneal surface.

Example 8 Effect of Intravitreal Administration of STC-1 in Rat Modelsof Retinal Degeneration

1.1. Rationale.

To determine whether intravitreal administration of STC-1 delaysphotoreceptor degeneration in vivo, two rodent models of retinaldegeneration were used. S334ter-3 rhodopsin transgenic rats were treatedat postnatal day 9 (P9) and again at P12 with intravitreal injections of1 μg STC-1. The rats were sacrificed at P19. Histologic analysisrevealed increased outer nuclear (ONL) thickness compared to uninjected(UI) controls. Mean ONL thicknesses of inferior (inf), superior (sup),and total retina were quantified as described previously in Lewin, etal., Nat. Med. Vol. 4, No. 8, pgs. 967-971 (1998). Total retina ONLthickness was increased significantly in STC-1 treated rats (n=4,p=0.018) (FIG. 23).

Additionally, the Royal College of Surgeons (RCS) rat model of retinaldegeneration was tested. As part of these studies, a gene expressionassay was developed that could be used in conjunction with histologicand functional analysis to detect photoreceptor rescue. Expression ofphotoreceptor specific genes was tested by quantitative real-time PCR(qRT-PCR) in retinas isolated from RCS rats at varying timepoints.Photoreceptor gene expression was shown to decrease over time in the RCSrat in rates comparable with the previously described decline of the ONLin LaVail, et al., Exp Eye Res., Vol. 21, No. 2, pgs. 167-192 (1975)(FIG. 24).

Utilizing this assay as a basis for quantification of photoreceptorviability, the hypothesis that intravitreal administration of STC-1would improve photoreceptor viability was tested. Rats received anintravitreal administration of 2.5 mg STC-1. Injections occurred at P21,the approximate time of initiation of ONL decline (LaVail, 1975). Therats were sacrificed, and tissue was collected at P40. Total RNA fromthe retinas of the rats was extracted using the RNeasy mini kit.(Qiagen). cDNA was generated by reverse transcription (Super Script III;Invitrogen) using 1 μg total RNA. Real time amplification was performedusing the Taq Man Universal PCR Master Mix (Applied Biosystems). An 18SrRNA probe (Taq Man) was used for normalization of gene expression. Geneexpression analysis showed significant increases in photoreceptor geneexpression in STC-1 treated animals compared to PBS injected controls(FIG. 25).

1.2. Design.

Intravitreal administration of STC-1 reduced photoreceptor degenerationin two rodent models of retinal degeneration. As described hereinbelow,the next set of experiments is directed to optimizing the dose, time ofadministration, and frequency of administration of STC-1 in the RCS rat.

1.2.1. Optimization of Dose of STC-1.

First, the dose of STC-1 which preserves photoreceptor viability mosteffectively (STC-1^(OptD)) is determined The experiments are carried outas summarized in Table 6. For this experiment, STC-1 is injected at P21and tissues are harvested at P40. The results are assessed on the basisof photoreceptor gene expression (see FIG. 24).

TABLE 6 Dose of STC-1. P21, postnatal day 21; P40, post-natal day 40.qRT-PCR, for photoreceptor genes as described in FIG. 24.; fellow eye,indicates the fellow (contralateral) eye is injected as an internalcontrol. Dose Injec- Termi- Evalua- No. Therapy (μg) tion nation tions(RCS Rat) STC-1 2.5 P21 P40 qRT-PCR 6 1.0 P21 P40 qRT-PCR 6 0.5 P21 P40qRT-PCR 6 0.1 P21 P40 qRT-PCR 6 0.05 P21 P40 qRT-PCR 6 PBS 0 P21 P40qRT-PCR fellow eye Milestone: Optimal dose of STC-1 = STC-1^(OptD).

In parallel Experiments, as outlined in Table 7 below, the optimal timeof administration will be defined and whether two injections are moreeffective than one will be determined.

TABLE 7 Day of injection and number of treatments. No. Injection Termi-Evalua- (RCS Therapy Dose 1^(st) inj. 2^(nd) inj. nation tions Rat)STC-1 STC-1^(OptD) P14 none P40 qRT-PCR 6 STC-1^(OptD) P14 P21 P40qRT-PCR 6 STC-1^(OptD) P21 P28 P40 qRT-PCR 6 STC-1^(OptD) P28 none P40qRT-PCR 6 STC-1^(OptD) P28 P35 P40 qRT-PCR 6 PBS 0 fellow eye P40qRT-PCR fellow eye Milestone: Optimal time of administration of STC-1 =STC-1^(OptT).

1.2.2. Histological and Functional Therapeutic Rescue by STC-1^(OptD)and STC-1^(OptT).

Histologic and functional tests are used to test the ability ofSTC-1^(OptD) and STC-1^(OptT) to rescue retinal degeneration compared toPBS injected controls. Fixed eyes are sent for quantification of ONLthickness as described in Lewin et al., 1998. Electroretinogram (ERG)analysis is performed as described previously in Ren et al., Exp EyeRes.; Vol. 70, No. 4, pgs. 467-473 (2000).

TABLE 8 Histologic and functional tests of photoreceptor rescue. Ter-No. Therapy Dose Injection(s) mination Evaluations (RCS Rat) STC-1STC-1^(OptD) STC-1^(OptT) P40 Histology 6 STC-1 STC-1^(OptD)STC-1^(OptT) P40 ERG 6 PBS 0 P40 fellow eye Milestone: Anatomic andfunctional measurements of the ability of STC-1 to rescue retinaldegeneration.

2.1. Rationale.

In order to determine whether cell therapy may provide greater rescue ofphotoreceptor viability than protein therapy alone, the hypothesis thatintravitreally administered human MSCs can survive in the rat vitreouscavity was tested. Vials of frozen passage one human mesenchymal stemcells (hMSCs) were obtained from the Center for the Preparation andDistribution of Adult Stem Cells (website accesible atmedicine.tamhsc.edu/irm/msc-distribution.html). Following 24-hourrecovery, the hMSCs were plated at a density of 100 cells/cm² andincubated at 37° C. in complete culture medium (CCM) with 16% fetalbovine serum (FBS) for 8 days until approximately 70% confluence wasreached. Passage three cells were used for all experiments.

An intravitreal injection of 1×10⁵ human MSCs was performed at postnatalday 21. Four days following injection the eye was enucleated and RNA wasextracted from the whole globe to evaluate how many human cells remainedin the eye cavity. Expression of human GAPDH (hGAPDH) was measured byqRT-PCR as hereinabove described, thereby providing an indication of anysurviving human cells in the rat eye. Based on the standard curvegenerated (FIG. 26, Left Panel), an estimated 10-20% of injected cellsremained viable for four days following injection (FIG. 26, RightPanel). Additionally, other secreted factors from MSCs including theanti-inflammatory protein TSG-6 (Lee, 2009), neurotrophic factors (Li,et al., Graefe's Arch. Clin. Exp. Ophthalmal., Vol. 247, No. 4, pgs.503-514 (2009)) such as CNTF, bDNF, or bFGF, or the retinal-protectiveprotein LIF (Bartosh, et al., Proc. Nat. Acad. Sci., Vol. 107, No. 31,pgs. 13724-13729 (2010); Joly, et al; J. Neurosci., Vol. 28, No. 51,pgs. 13765-13774 (2008)) may act in conjunction with STC-1 to preservephotoreceptor viability.

MSCs in standard culture conditions express relatively low levels oftherapeutic proteins unless stimulated in culture or activated in vivoby injury signals from the host (Lee, 2009; Bartosh, 2010). A recentreport from our laboratory demonstrated that culturing MSCs as 3Dspheroids activates the cells to produce large amounts of therapeuticmolecules including STC-1 (Bartosh, 2010). Compared to standard culturepreparations of MSCs, culture of the cells as 3D spheroids of 25,000cells (Sph 25K) enhanced secretion of STC-1 about 20-fold. Spheroids canbe dissociated into MSC spheroid dissociated cells (Sph 25 k DC) whichretain the ability to secrete high levels of STC-1 compared to monolayerMSCs (Adh High) (FIG. 27).

2.1.1. Optimization of the Dose of Sph 25 k DCs.

First, the dose of Sph 25 k DCs which preserves photoreceptor viability(Sph 25 k DC^(OptD)) most effectively is determined. The experiments arecarried out as summarized in Table 9. For this experiment, Sph 25 kbased on photoreceptor gene expression (see FIG. 24) are injected.

TABLE 9 Dose of Sph 25 k DCs. qRT-PCR analysis for photoreceptor genesas described in Fig. 24. Dose Injec- Termina- Evalua- No. Therapy (×10³cells) tion tion tions (RCS Rat) Sph 25 k DCs 100 P21 P40 qRT-PCR 6 50P21 P40 qRT-PCR 6 10 P21 P40 qRT-PCR 6 MSCs 100 P21 P40 qRT-PCR 6 50 P21P40 qRT-PCR 6 10 P21 P40 qRT-PCR 6 Fbs 100 P21 P40 qRT-PCR 6 50 P21 P40qRT-PCR 6 10 P21 P40 qRT-PCR 6 PBS 0 P21 P40 qRT-PCR fellow eyeMilestone: Optimal dose of Sph 25 k DCs = Sph 25 k DC^(OptD).

2.1.2. Optimization of the Administration Time and Frequency of Sph 25 kDCs.

In parallel experiments, carried out as summarized in Table 10 below,the optimal time of administration is defined and whether two injectionsare more effective than one is determined.

TABLE 10 Time and frequency of administration of Sph 25k DCs. No.Injection Termi- Evalua- (RCS Therapy Dose 1^(st) inj. 2^(nd) inj.nation tions Rat) Sph 25k SPH 25k P14 none P40 qRT-PCR 6 DCs DC^(OptD)SPH 25k P14 P21 P40 qRT-PCR 6 DC^(OptD) SPH 25k P21 P28 P40 qRT-PCR 6DC^(OptD) SPH 25k P28 none P40 qRT-PCR 6 DC^(OptD) SPH 25k P28 P35 P40qRT-PCR 6 DC^(OptD) PBS 0 fellow P40 qRT-PCR fellow eye eye Milestone:Optimal time of administration of Sph 25k DCs = Sph 25k DC^(OptT).

2.1.3. Histological and Functional Therapeutic Effects of Sph 25 kDC^(OptD) and Sph 25 k DC^(OptT).

Histologic and functional tests carried out as described in Table 11below are used to test the ability of Sph 25 k DC^(OptD) and Sph 25 kDC^(OptT) to rescue retinal degeneration compared to PBS control.

TABLE 11 Histologic and functional tests of photoreceptor rescue. No.Ter- Eval- (RCS Therapy Dose Injection mination uations Rat) Sph 25 kDCs Sph 25 k DC^(OPtD) Sph 25 k P40 Histology 6 DC^(OptT) Sph 25 kDC^(OptD) Sph 25 k P40 ERG 6 DC^(OptT) PBS 0 P40 Histo/ERG fellow eyeMilestone: Anatomic and functional evidence of the rescue effects of Sph25 k DCs.

3.1 Rationale.

It has been proposed that patients with RP undergo cone photoreceptordeath due to oxidative damage following rod photoreceptor death (Shen,2005; Usui, Mol. Ther., Vol. 17, No. 5, pgs. 778-786 (2009)). Increasedlevels of oxygen have been observed in rodent models of RP including theRCS rat as photoreceptor degeneration occurs (Yu, 2004; Yu, et al.,Invest. Ophthalmol. Vis. Sci., Vol. 41, No. 12 pgs. 3999-4006 (2000)).As a result of increased levels of oxygen, oxidative damage tophotoreceptors has been observed both in small (Komeima, 2006) and large(Shen, 2005) animal models of RP. Further evidence includes studies thatdemonstrate antioxidant therapy slows photoreceptor death in animalmodels of RP (Komeima, 2007). The hypothesis was tested initially in anin vitro model of oxidative RPE injury. Human RPE cells from cell lineARPE-19 (ATCC Catalog No. CRL-2302), were damaged with 450 μM hydrogenperoxide as described previously in Kim, et al., Korean J. Ophthalmol.,Vol. 17, No. 1, pgs. 19-28 (2003). As summarized in Table 12 below, onehour after injury, the cells were treated with 250 ng/ml STC-1, or witha vehicle (control).

TABLE 12 In vitro study using STC-1 treatment of hydrogenperoxide-damaged ARPE-19. Therapy Dose Assays STC-1 250 ng/ml TUNEL,mito. potential, lactate production, qRT-PCR Vehicle TUNEL, mito.potential, lactate production, qRT-PCR

The cells, following treatment, were evaluated for expression of apro-apoptotic gene (caspase 3/7), cell death (Annexin V & PI staining ofcells) and improved cell viability (increased activity of themitochondrial enzyme MTT). Detection of caspase activity was performedas described in Sharma, et al., Invest. Ophthalmol. Vis. Sci., Vol. 49,No. 11, pgs. 5111-5117 (2008), annexin/PI quantification as described inBartosh, et al., Proc. Nat. Acad. Sci., Vol. 107, No. 31, pgs.13724-13729 (2010), and MTT conversion was measured as describedpreviously in Mester, et al., J. Mol. Neurosci. (2010).

Following injury, it was observed that treatment with STC-1 reducedapoptosis and improved cell viability compared to vehicle controls (FIG.28).

Additionally, following an intravitreal injection of 2.5 μg STC-1 in theRCS rat at P21, gene expression of BAX, a transcript that encodes apro-apoptotic protein, is reduced significantly in the RCS retina at P40(FIG. 29) as assessed by qRT-PCR. Therefore, these results areconsistent with the idea that STC-1 inhibits apoptosis in the RCS ratretina. The hypothesis that the results are because of the ability ofSTC-1 to reduce reactive oxygen species by uncoupling oxidativephosphorylation then is tested.

3.2. Design.

In vitro experiments are carried out as summarized in Table 9. In vivoexperiments are carried out as summarized in Table 10. First, the invitro model of hydrogen peroxide induced ARPE-19 injury (Kim, 2003) isused. Apoptosis in vitro is evaluated using TUNEL stain, lactateproduction (Tanito, Invest. Ophthalmol. Vis. Sci., Vol. 46, No. 3, pgs.979-987 (2005)), and qRT-PCR. The level of reactive oxygen species, orROS, is evaluated using measurements of mitochondrial potential. Changesin UCP2 with qRT-PCR also is evaluated.

For in vivo studies, apoptosis in the retina of RCS rats is evaluatedusing TUNEL stain (Mizukoshi, Exp. Eye Res., Vol. 91, No. 3, pgs.353-361 (2010)) and qRT-PCR. In addition, levels of ROS are evaluated bymeasuring tissue aconitase activity as described previously in Tarpey,et al., Am. J. Physiol. Regul. Integr. Comp. Physiol., Vol. 286, No. 3,pgs. 431-444 (2004).

TABLE 10 In vivo study using intravitreal administration of STC-1 in theRCS rat. Ter- No. Therapy Dose Injection(s) mination Assays (RCS Rats)STC-1 STC-1^(OptD) STC-1^(OptI) P40 TUNEL, 6 aconitase, qRT-PCR VehicleP40 TUNEL, 6 aconitase, qRT-PCR

Example 9, Effect of Topical TSG-6 on Corneal Inflammation

Fifteen mice were anesthetized by isoflurane inhalation. In order tocreate a chemical burn to the cornea, 100% ethanol was applied to thewhole cornea including the limbus for 30 seconds, followed by rinsingwith 10 ml of balanced salt solution. The whole cornea and limbalepithelium then were scraped mechanically using a surgical blade. (SeeOh et al., Proc. Nat. Acad. of Sci., Vol. 107, No. 39, pgs. 16875-16880(Sep. 28, 2010)). Two mice served as controls Immediately thereafter, 5μl of PBS were administered intravenously or intraperitoneally to 10mice, and 2 μg/5 μl of TSG-6 were applied topically to the cornealsurfaces of 5 mice. The eyelids of the mice then were closed with one8-0 silk suture at the lateral third of the lid margin.

Three days later, the mice were killed and the corneas were excised. Thecorneas then were sectioned into small pieces and lysed in 150 μl oftissue extraction reagent containing protease inhibitors (Invitrogen).The samples then were sonicated on ice and centrifuged twice (15,000×gat 4° C. for 20 minutes). The supernatants were assayed with commercialELISA kits for myeloperoxidase (MPO) (MPO ELISA kit, Hy Cult Biotech).

As shown in FIG. 30, topical administration of TSG-6 suppressed cornealinflammation more effectively than the controls.

Example 10

Sterile inflammation now is recognized to play a key role in manydiseases that include myocardial infarction, stroke, Alzheimer'sdisease, and atherosclerosis (Chen et al., Nat. Rev. Immunol., Vol. 10,No. 12, pgs. 826-837 (2010); Rock et al., Ann. Rev Immunol, Vol. 28,pgs. 321-342 (2010); Spite et al., Circ. Res., Vol. 107, No. 10, pgs.1170-1184 (2010)). The molecular and cellular responses of sterileinflammation include over 20 nonmicrobial endogenous stimuli referred toas damage-associated molecular patterns (DAMPs) which signal throughpattern recognition receptors (PRRs) on resident macrophages thatactivate at least three intracellular pathways to upregulate theexpression of pro-inflammatory cytokines. In spite of the intenseinterest in the field, a series of important questions remainunanswered, including whether some DAMPs identified from roles in vitroplay important roles in vivo, whether some DAMPS play redundant roles,and whether different tissues used different DAMPs (Matzinger, Nat.Immunol., Vol. 8, No. 1, pgs. 11-13 (2007)).

The cornea is an attractive model system to investigate sterileinflammation because it is accessible readily to experimentalmanipulations in vivo and in vitro. Moreover, sterile inflammationoccurs in diseases of the cornea that include limbal stem celldeficiency, chemical burns, and allergic or autoimmune keratitis(Wagner, Surv. Ophthalmol., Vol. 41, No. 4, pgs. 275-313 (1997);Krachmer, Cornea, 2^(nd). Ed., Vol. 1, pgs 1179-1308). To examine thetemporal sequence and stimuli for sterile inflammation, a model was usedin which the cornea was injured by exposure to alcohol followed byscraping to remove the epithelium of the cornea and limbus that containsstem cells. It was observed that the injury provoked two distinct phasesof neutrophil infiltration: A small initial Phase I that began in 15 mmand reached a plateau between 4 to 8 hours and a much larger secondPhase II that peaked at 24 hours to 48 hours. Analysis of the two phasesdemonstrated that Phase I was stimulated by the neuropeptidesecretoneurin and perhaps other signals. The second, more massive PhaseII of neutrophil infiltration was simulated by a small heat shockprotein, HSPB4, that was synthesized and released in injured keratocytesof the corneal stroma and that acted as a DAMP to activate residentmacrophages.

Methods

Animals. Lewis rats (LEW/Crl) were purchased from Charles RiverLaboratory (Wilmington, Mass.). HSPB4 knockout mice (Cryaa⁻¹⁻) weregenerated originally at the National Eye Institute by targeted genedisruption and were maintained in the 129 S6/SvEvTac background (Bradyet al. Proc. Nat. Acad. Sci., Vol. 94, No. 3, pgs. 884-889 (1997).129S6/SvEvTac, C57BL/6 (C57BL/6J) and CD44 knockout mice (CD44h⁻¹⁻;B6.Cg-Cd44tm1Hbg/J) were purchased from the Jackson Laboratory (BarHarbor, Me.). All animals were used under a protocol approved by theInstitutional Animal Care and Use Committee of Texas A&M Health ScienceCenter College of Medicine.

Animal Models of Injury and Treatment

Injury was created by applying 100% ethanol to the whole corneaincluding the limbus for 30 seconds followed by rinsing with 10 ml ofbalanced salt solution. Then, the whole corneal and limbal epitheliumwas scraped mechanically using a surgical blade.

For injection of the recombinant human SN (PolyPeptide Laboratories,HiHerod, Denmark) or HSPB4 (Enzo Life Sciences, Plymouth Meeting, Pa.),2 μl of the proteins in PBS (0.2 ng SN in 2 μl PBS or 100 ng HSPB4 in 2μl PBS; the amount of SN or HSPB4 detected in the cornea at 15 min afterinjury) were injected using a 32 gauge needle into the corneal stromanear the temporal limbus. The proteins were purified by endotoxinbinding columns and sterilized prior to use according to themanufacturer's instructions (EndoClear, blue; Hycult Biotech Inc.Plymouth Meeting, Pa.), and tested to be free of detectable levels ofGram-negative bacterial endotoxins (<0.01 EU/ml) or proteins (1 ng/ml)using the Limulus amoebocyte lysate kit (Hycult Biotech Inc.) and E.coli HCP ELISA kit (Cygnus Technologies, Southport, N.C.).

For macrophage depletion, either 100 μl of clodronate-encapsulatedliposome (5 mg clodronate per ml suspension; Encapsula Nano Sciences,Nashville, Tenn.) or the same volume of PBS-encapsulated liposomes wereinjected subconjunctivally near the limbus on day −2 (2 days beforeinjury) and on day 0 (day of injury). The injection was dispensed overfour quadrants (25 μl each) so that a circular bleb around the corneawas formed.

For blocking the release of SN from nerve endings, 20 nl of 2 mMdiltiazem solution in isotonic saline (Sigma-Aldrich, St. Louis, Mo.)was applied topically to the cornea 15 min prior to injury (Gonzalez etal., Invest. Ophthalmol. Vis. Sci., Vol. 34, No. 12, pgs. 3329-3335(1993)). For blocking HSPB4 in the cornea, either mouse monoclonal orrabbit polyclonal antibodies to rat HSPB4 (10 μg or 50 μg in 100 nl PBS;Abcam, Cambridge, Mass.) were injected subconjunctivally near the limbusright before the injury. The same concentration of isotype IgG also wasinjected as control.

To evaluate the effect of TLR2 inhibition in the injured cornea, rhTSG-6(2 ng in 5 μL of PBS; R&D Systems, Minneapolis, Minn.) or the samevolume of PBS was injected into the anterior chamber of the rat eyeimmediately after injury.

Cells and Cell Lines

Murine macrophages (RAW 264.7) were obtained from ATCC (Rockville, Md.).Human embryonic kidney (HEK) 293 cells transfected with vectorsexpressing human TLR2 or TLR4 plus a vector expressing an alkalinephosphatase reporter gene under the control of an inducible NF-kBpromoter were purchased from InvivoGen (HEK-Blue™-hTLR2 andHEK-Blue™-hTLR4; San Diego, Calif.). A control cell line not expressingeither TLR2 or TLR4 also was obtained and used (HEK-Blue™—Null1). Thestable cell line expressing human CD44 (Origene, Rockville, Md.) orPcDNA 3.1 control vector (Invitrogen) was generated. The primary humankeratocytes were obtained from ScienCell (Carlsbad, Calif.) and used atpassage 5.

Cell Injury Induction

To see the effects of injured cells in vitro, necrotic corneal tissueextracts were prepared. Rat corneas were homogenized in PBS (100 μl perone cornea) using a motor-driven homogenizer followed by fivefreeze-thaw cycles and 37° C. for 5 hours (Chen, et al., Nat. Med., Vol.13, No. 7, pgs. 851-856 (2007)). After centrifugation at 12000 rpm for 5min, the supernatants were prepared as necrotic extracts. Some ofnecrotic extracts were heat-treated (100° C., 20 min) Necrotic extractswere incubated with the cells in culture at a 1:10 dilution for 2 hours.To evaluate the effect of HSPB4, either antibodies to HSPB4 (10 ng or 50ng) or isotype IgG antibodies also were added to the cultures.

To evaluate the effects of sHSPs and SN, either crystallins (HSPB4,HSPB5, PB crystallin; 0.001 to 10 ng/ml) or SN (0.1 to 10 ng/ml) wereadded to cultures and the cultures were incubated for 2 hours. To seethe effect of TSG-6, 100 ng/ml or 500 ng/ml of rhTSG-6 were added to thecultures. To rule out the possibility of bacterial or LPS contaminationof sHSPs, all in vitro experiments were done in the presence ofpolymyxin B (10 μg/ml) for neutralization of LPS. Moreover, foradditional control experiments, sHSPs denatured by heat (100° C., 20min) were used in parallel sets of experiments. To see whetherkeratocytes express sHSPs in response to injury, either necroticextracts or H₂O₂ (100 to 500 nM) were added to the cultures ofkeratocytes.

Measurement of the Myeloperoxidase Amount in the Cornea

For a quantitative measure of neutrophil infiltration, the corneas wereassayed for the myeloperoxidase (MPO) concentration (Rat MPO EL1SA kit;HyCult biotech) as reported previously (Oh et al., Proc. Nat. Acad.Sci., Vol 101, No. 39, pgs. 16875-16880 (2010)). For protein extraction,the cornea was cut into small pieces and lysed in 150 μl of tissueextraction reagent (Invitrogen, Carlsbad, Calif.) containing proteaseinhibitor cocktail (Roche, Indianapolis, Ind.). The samples weresonicated on ice using an ultrasound sonicator. After centrifugation at12,000 rpm at 4° C. for 20 min, the cleared supernatant was collectedand assayed for levels of MPO.

Microarrays

RNA target for microarrays was prepared using the 3′ IVT Express Kit(Affymetrix) according to manufacturer's instructions. Briefly, 200 ngof total RNA was used to synthesize first strand cDNA. The cDNA then wasconverted into double-stranded cDNA and used in in vitro transcriptionto synthesize biotinylated cRNA. The cRNA was purified with magneticbeads, fragmented, and 12.5 μg were used in the hybridization ontoRG-230 2.0 arrays. The arrays were stained, washed, and scanned forfluorescence. Microarray data was normalized and analyzed using thePartek Genomics Suite 6.4 (Partek) and dChip software. For comparativeanalysis, data were filtered based on fold changes of 2 or more (eitherup- or down-regulated). For the hierarchical clustering analysis datawere filtered using a coefficient of variation higher than 0.6 and apresence call of at least 33%. The expression levels of the filteredgenes were standardized and used in hierarchical clustering. A total 6clusters were selected in each hierarchical clustering on the similarlevel of hierarchy and studied for enriched Gene Ontology tags based onhypergeometric distribution.

Real-Time RT-PCR

Total RNA from the cornea or the cells was extracted (RNeasy Mini kit;Qiagen, Valencia, Calif.) and used to synthesize double-stranded cDNA byreverse transcription (SuperScript III; Invitrogen). Real-timeamplification was performed (Taqman Universal PCR Master Mix AppliedBiosystems, Carlsbad, Calif.) and analyzed on an automated instrument(7900HT Fast Real-Time PCR System; Applied Biosystems). PCR probe setswere purchased commercially (Taqman Gene Expression Assay Kits, AppliedBiosystems). For assays, reactions were incubated at 50° C. for 2 min,95° C. for 10 min, and then 40 cycles at 95° C. for 15 sec followed by60° C. for 1 min. For normalization of gene expression, 18S rRNA probe(Taqman Gene Expression Assays ID, Hs03003631_g1) was used as internalcontrol. The threshold cycle (Ct) was used to detect the increase in thesignal associated with an exponential growth of PCR products during thelog-linear phase. The expression of molecules was calculated using thealgorithm 2^(−ΔΔCt).

Western Blot

Clear lysates prepared as described above were measured for proteinconcentration, and a total of 10 μg protein was fractionated by SDS-PAGEon 10% bis-tris gel (Invitrogen), transferred to nitrocellulose membrane(Invitrogen), and blotted with antibodies against SN (PhoenixPharmaceuticals, Burlingame, Calif.) or HSPB4 (Abcam).

ELISAs

Protein was extracted from the cornea as described above, and wasassayed for levels of pro-inflammatory cytokines and chemokines withcommercial ELISA kits for IL-6, IL-1β, and CXCL1/CINC-1 (Quantikine kit;R&D Systems), and for CCL2/MCP-1 (Immunoassay Kit; Invitrogen). ForHSPB4 measurement, mouse monoclonal anti-rat antibody to HSPB4 (Abcam)was used as a capture antibody (4 μg/ml) and rabbit polyclonal anti-ratantibody to HSPB4 (Abcam) as a secondary antibody (400 ng/ml).

Release of HSPB4 in Injured Cornea

To measure the amount of HSPB4 released from the cornea after injury,the corneas of rats were harvested immediately after the injury andcultured at 37° C. with 5% CO₂ for 12 hours. Every two hours, theculture medium was changed and the concentration of HSPB4 in conditionedmedium during each time frame was measured by ELISA.

Histopathology

The cornea was excised after the rat was sacrificed and fixed in 10%paraformaldehyde. The cornea was cut into 4 μm sections and stained withthe hematoxylin-eosin (H&E) or subjected to immunohistochemistry. Theformalin-fixed corneal section was deparaffinized with ethanol andantigen was retrieved using an epitope retrieval solution (IHC WORLD,Woodstock, Md.). The rabbit polyclonal anti-rat antibody to neutrophilelastase (1:200, Abcam), the mouse monoclonal anti-rat antibody tosecretogranin II (1:200, Abcam), or the mouse monoclonal anti-ratantibody to HSPB4 (1:200, Abcam) were used as primary antibodies, andthe anti-rabbit IgG (1:5000, Abcam) or the anti-mouse IgG (1:5000,Abcam) as secondary antibodies. The DAPI solution (VECTASHIELD MountingMedium; Burlingame, Calif.) was used as counterstaining

Aconitase Activity Assay

To evaluate the oxidative damage in the cornea by injury (Ma et al.,Biochem Biophys. Acta, Vol. 1790, No. 10. pgs. 1021-1029 (2009), loss ofaconitase activity in the corneal lysates was measured using anaconitase assay kit according to the manufacturer's protocol (CaymanChemical Company, Ann Arbor, Mich.)

NF-kB Translocation Assays

About 1×10⁵ mouse macrophages were plated in 8 well chamber slides(Lab-Tek II Chamber Slide; Nalge Nunc, Rochester, N.Y.) and incubatedfor 1 hour in 0.2 mL of 2% FBS in α-MEM with or without 10 μg/mL HSPB4.The cells were washed twice with PBS by centrifugation and were fixedwith 100% methanol for 5 min. The cells were washed with PBS and blockedwith Image-iT™ FX Signal Enhancer (Invitrogen). The cells then wereincubated with 1 mg/mL of anti NF-kB p65 antibody (Abcam) in blockingbuffer (5% BSA in PBS) overnight at 4° C. The samples were incubated for1 hour with a 1:2000 dilution of the secondary antibody of anti-rabbitIgG (Alexa Fluor® 488 goat; Invitrogen). The DAPI solution was used tostain the cell nuclei. The slides were visualized with fluorescentmicroscopy using an upright microscope (Eclipse 80i, Nikon, Melville,N.Y.)

Statistical Analysis

Comparisons of parameters among the groups were made by the Student's ttest, non-parametric Mann-Whitney test or Pearson's correlation testusing SPSS software (SPSS 12.0). Differences were considered significantat p<0.05.

Results

Two Phases of Neutrophil Infiltration after Sterile Injury to the Cornea

The corneas of Lewis rats, were injured by exposing them to 100% ethanolfor 30 seconds and scraping of the cornea and limbus to remove both theepithehum and stem cells found in the limbus. As described previously(Oh et. al, Proc. Nat. Acad. Sci., Vol. 107, No. 34, pgs. 16875-16880(2010)), neutrophil infiltration was monitored by assays formyeloperoxidase (MPO) that is stored within neutrophil granules andreleased by activation of the cells (Borregard et al., Blood, Vol. 89,pgs. 3503-3521 (1997)). The neutrophil infiltration occurred in the twophases. There was a small initial phase that began within about 15 min,and reached a plateau level at 4 to 8 hours (Phase I in FIG. 31A). Afterthe plateau, a much larger infiltration of neutrophils followed andreached a maximum at 24 to 48 hours (Phase II in FIG. 31A). Theneutrophils then gradually disappeared in a recovery phase over 48 hoursto 7 days.

Search for Candidate Signals for Phase I and Phase II

As a strategy to identify candidate signals that initiated the twophases, microarrays were used to survey the response of the cornea toinjury. Based on the temporal pattern of gene expression, the genes wereclassified into three groups (FIGS. 31B, FIG. 31D). About 842 Group Agenes were up-regulated, about 108 Group B genes were up-regulated, andabout 307 Group C genes were up-regulated (FIG. 31D). Focus was directedto the Group A genes, because they were expressed earlier and thereforemore likely to include stimuli for Phase I and II (FIGS. 31B and C).Most of the Group B genes were the molecules related to apoptosis/deathand defense response (See Table 11 below, FIG. 31D). Most of the Group Cmolecules were pro-inflammatory chemokines and cytokines (See Table 11below, FIG. 31D, FIG. 32M), and therefore were likely to be the genesthat peaked late in the inflammatory responses. Most of the Group Agenes were genes for nerve/neurotransmission-related and structuralproteins of the eye (See Table 11 below.) From the category of Group Agenes, the following were selected as attractive candidates forinflammatory signals: the neuropeptide secretoneurin (SN) because it wasshown previously to activate chemotaxic migration and transendothelialextravasation of blood cells (Helle, Regul Pept., Vol. 165, No. 1, pgs.45-51 (2010); Taupenot et al., N. Engl. J. Med., Vol. 348, No. 12, pgs.1134-1149 (2003)), and two small heat shock proteins (HSPB4 and HSPB5),because some heat shock proteins were previously shown to act as DAMPs(Joly et al., J. Innate Immun, Vol. 2, No. 3, pgs. 238-247 (2010); VanWijk et al., J. Leukoc. Biol., Vol. 88, No. 3, pgs. 431-434 (2010);Quintana et al., J. Immunol., Vol. 175, No. 5, pgs. 2777-2782 (2005);Asea et al., Nat. Med., Vol. 6, No. 4, pgs. 435-442 (2000)).

TABLE 14 Microarray analysis of gene expression profiles in the corneaat 4 hours and 24 hours after injury. The top 20 transcriptsup-regulated by injury were shown in each group (Group A, B, and C).Change (x-Fold) Gene Injured (4 h)/ Injured (24 h)/ Gene Title symbolProbe set con con Group A crystallin, alpha A Cryaa 1370279_at 19.4681.620 crystallin, beta B1 Crybb1 1369985_at 18.541 1.062 crystallin,gamma C Crygc 1370292_a_at 17.911 1.112 secretogranin II ScgII1368044_at 15.847 1.033 claudin 2 Cldn2 1375933_at 15.246 1.033crystallin, beta A1 Crybal 1371408_at 14.628 −3.593 crystallin, gamma BCrygb 1371413_x_at 14.311 1.025 crystallin, gamma D Crygd 136770._at13.957 1.054 synaptosomal-associated Snap25 1387073_at 13.199 1.580protein 25 Galectin-related inter- Grifin 1386936_at 12.710 −1.129 fiberprotein solute carrier family 6 Slc6a1 1368170_at 12.359 −1.704(neurotransmitter transporter, GABA), member 1 crystallin, gamma S Crygs1388435_at 11.969 −1.096 Calbindin Calb1 1370201_at 11.542 −1.184crystallin, beta A2 Cryba2 1388385_at 10.349 −1.497 crystallin, beta B2Crybb2 1367684_at 9.918 −1.738 retinol binding protein 3, Rbp31376777_at 9.789 −1.434 interstitial collagen, type II, alpha 1 Col2a11387767_a_at 8.615 −1.224 phosphodiesterase 6A, Pde6a 1393426_at 8.500−1.011 cGMP-specific, rod, alpha complexin 3 Cplx3 1384779_at 8.061−1.099 crystallin, alpha B Cryab 1370026_at 7.197 1.812 crystallin, betaA4 Cryba4 1367608_at 7.168 −1.749 Group B interleukin 6 Il6 1369191_at340.974 293.81 colony stimulating factor Csf3 1369529_at 70.050 62.614 3(granulocyte) matrix metallopeptidase Mmp13 1388204_at 30.118 28.285 13prostaglandin E synthase Ptges 1368014_at 19.449 15.069 metallothionein2A Mt2A 1388271_at 17.356 13.067 superoxide dismutase 2, Sod2 1370173_at16.915 11.803 mitochondrial interleukin I alpha Illa 1371170_a_at 14.6375.275 prostaglandin- Ptgs2 1368527_at 10.861 8.108 endoperoxide synthase2 metallothionein 1a Mtla 1371237_a_at 9.483 6.815 lipocalin2 Lcn21387011_at 9.013 9.387 immediate early response Ier3 1388587_at 5.5525.781 3 prostaglandin E synthase Ptges 1368015_at 5.433 5.051 sixtransmembrane Steapl 1393706_at 5.221 4.467 epithelial antigen of theprostate 1 cAMP responsive element Crem 1393550_at 5.213 4.998 modulatortransferrin receptor Tfrc 1388750_at 5.182 5.061 mitogen-activatedprotein Map3k8 1369393_at 4.935 5.302 kinase 8 B-cell translocation geneBtg2 1386994_at 4.931 4.677 2, anti-proliferative runt relatedtranscription Runx 1 1368914_at 4.924 3.913 factor 1 similar to F-boxonly RGD1563982 1375041_at 4.717 3.358 protein 27 growth arrest, DNA-Gadd45a 1368947_at 4.026 3.756 damage-inducible, alpha Group C secretoryleukocyte Slpi 1367998_at 54.415 533.847 peptide chemokine (C—X—C motif)Cxcl2 1368760_at 52.728 404.344 ligand 2 S100 calcium binding Sl00a91387125_at 56.114 207.701 protein A9 interleukin 1 beta Il1b 1398256_at33.522 198.673 chemokine (C—C motif) Ccl7 1379935_at 40.595 159.69ligand 7 S100 calcium binding Sl00a8 1368494_at 33.206 155.69 protein A8chemokine (C—X—C motif) Cxcl3 1370633_at 7.895 128.417 ligand 3chemokine (C—C motif) Cc13 1369815_at 6.493 95.538 ligand 3 interleukin1 receptor, Il1r2 1387180_at 24.159 60.257 type II chemokine (C—X—Cmotif) Cxcl3 1370634_x_at 4.934 59.166 ligand 3 interleukin I alpha Il1a1368592_at 2.953 54.174 Fc fragment of IgG, low Fcgr2a 1367850_at 8.70841.195 affinity Ila. receptor Cd53 molecule Cd53 1368518_at 6.748 31.988chemokine (C—C motif) Ccr1 1370083_at 6.359 28.512 receptor I chemokine(C—X—C motif) Cxcl3 1388032_a_at 2.331 24.419 ligand 3 colonystimulating factor Cst3r 1386009_at 8.094 21.987 3 receptor Fc fragmentof IgG, high FcgrIa 1393038_at 3.611 21.032 affinity Ia, receptorlaminin, gamma 2 Lamc2 1379340_at 5.817 20.577 immunoglobulin lgsf61387687_at 2.915 20.148 superfamily, member 6 complement component 3 C31368000_at 6.292 15.602 Symbols: Injured (4 h)/con, cornea at 4 hoursafter injury vs. cornea right after injury; Injured (24 h)/con, corneaat 24 hours after injury vs. cornea right after injury; ‘−’ meansdown-regulation. The values are the results from collective samples of n= 4 per each group.SN as a Candidate for Phase I and HSPB4 for Phase II

Data on the time course of expression were consistent with SN serving asan initiating signal for Phase I. SN was not detected in extracts ofuninjured cornea, but appeared both in corneal extracts and the serum ofthe rats within 0.25 hour of the injury (FIGS. 32A and B). The levels ofSN in extracts of injured corneas and the serum decreased at 0.5 hourand then increased apparently as a result of increased expression of thegene (FIGS. 32A-D). In contrast, there were little changes in theexpression of genes for substance P and calcitonin gene-related peptide(CGRP), two other neuropeptides known to be expressed in the cornea(FIG. 32D) (Troger et al., Brain Res. Rev., Vol. 53, No. 1, pgs. 39-62(2007)).

Similar data on the time course of expression were consistent with HSPB4serving as a stimulus for Phase II. Uninjured cornea contained lowlevels of HSPB4 protein, but the amount increased beginning after 0.5hour of the injury and reached a peak at about 4 hours (FIGS. 32E andF). A similar time course was observed in the release of HSPB4 into themedium in experiments in which corneas were injured in vivo and thenincubated ex vivo (FIG. 32G). As expected from the microarray data,there was increased expression of mRNAs for HSPB4 and related genes fromGroup A (FIG. 32I); however, the increases in expression of HSPB4 weredelayed compared to the increase in the mRNA for SN (compare FIGS. 32Dand I). Immunohistochemistry of injured cornea was consistent with theresults. The SN-immunoreactive nerves were increased in the cornea at 2hours following injury (FIG. 32C). The expression of HSPB4 was increasedat about 4 hours following injury (FIG. 32H).

In addition, the expressions of SN and HSPB4 were dependent on theseverity of injury. The concentrations of both the proteins and mRNAswere higher in the cornea or serum following severe injury (30 secethanol and scraping) compared to mild injury (15 sec ethanol andscraping) (FIG. 33F).

Keratocytes from the Corneal Stroma Synthesized HSBP4 in Response toInjury

To define the cellular origin of HSPB4 in the injured cornea,keratocytes that are fibroblast-like cells from the corneal stroma wereincubated with extracts of the cornea that were made necrotic byrepeated freezing and thawing. The necrotic extracts induced theexpression of HSPB4 in the keratocytes (FIG. 32J). The necrotic extractsdid not increase significantly the expression of a second small heatshock protein HSPB5 or a third Group A gene, βB crystallin. Most sterileinflammations produce increases in reactive-oxygen species (ROS)(Kolnitzer et al., Ann. N.Y. Acad. Sci., Vol. 1203, pgs 45-52 (2010);Martinon, Eur. J. Immunol., Vol. 40, No. 3, pgs. 616-619 (2010)). Assaysof injured cornea demonstrated a rapid decrease in aconitase activity, areflection of an increase in ROS (FIG. 32K). As expected, incubation ofkeratocytes with H₂O₂ to increase ROS also produced increased expressionof HSPB4 (FIG. 32L).

Recombinant SN Reproduced Phase I and Recombinant HSPB4 Reproduced PhaseII

Injection of recombinant SN into the stroma of the cornea stimulated theneutrophil infiltration of Phase C (FIG. 33A). The effect was negatedpartially by topical application of a calcium blocker (Diltiazem) (FIG.33D) that inhibits release of neuropeptides (Gonzalez et al, Invest.Ophthalmol. Vis. Sci., Vol. 34, No. 12, pgs. 3329-3335 (1993). Theresults therefore indicated that SN served as a major stimulus for PhaseI, but they did not exclude the possibility that it acted in concertwith other signals released by the injured cornea.

Injection of recombinant HSPB4 that was pyrogen-free (see Methods)stimulated the neutrophil infiltration of both Phase I and Phase II(FIGS. 33B and C). Reproduction of Phase I was explained apparently bythe protein being injected earlier than it appears in the tissue afterinjury to the cornea (FIGS. 32E-H). Injection of recombinant HSPB4 intoone region of the cornea also reproduced the opacity produced by sterileinflammation (FIG. 33C). The role of HSPB4 was confirmed by experimentsin which antibodies to the protein were injected into the corneal stromaimmediately after the injury (FIG. 33E). The antibodies to HSPB4inhibited significantly the Phase II inflammatory response in the corneaafter the injury. Also, the neutrophil infiltration of Phase II in thecornea after injury was decreased significantly in the corneas of HSPB4knockout mice, compared to wild-type controls (FIG. 33F).

HSPB4 Activated Resident Macrophages through TLR2/NF-kB Signaling

To identify the cells that responded to HSPB4 in the cornea, residentmacrophages were depleted from the corneas of Lewis rats by injectingclodronate liposomes subconjunctivally, and then recombinant HSPB4 wasinjected into the corneal stromas. Macrophage depletion was confirmedwith immunostaining for macrophage-specific markers CD68 and CD11b (FIG.33G). The protein did not reproduce Phase II in rats in whichmacrophages were depleted (FIG. 34A), suggesting that the effects ofHSPB4 were dependent on the presence of resident macrophages. Similarly,the inflammation was decreased markedly in the cornea afterchemical/mechanical injury when resident macrophages were depleted priorto an injury (FIG. 34L), indicating a crucial role of residentmacrophages in sterile injury-induced inflammation of the cornea. Inparallel experiments, it was observed that neutrophil infiltration ofPhase II, but not Phase I, was suppressed significantly by anintraocular injection of the anti-inflammatory protein, TSG-6 (FIG.34B), that inhibits TLR2/NF-kB signaling in macrophages (Choi, Blood, inpress; Lesley et al., J. Biol. Chem., Vol. 279, pgs. 25745-25754(2004)). Also, TSG-6 suppressed significantly the Phase II inflammatoryresponse caused by HSPB4 injection to the corneal stroma (FIG. 34C). Theresults suggested therefore that HSPB4 signaled Phase II by activatingTLR2/NF-kB signaling in resident macrophages.

To test the effects of HSPB4 on macrophages, macrophages were incubatedwith extracts of necrotic corneas. The extracts increased the expressionof the pro-inflammatory cytokines IL-1α, IL-1β, and IL-6 (FIG. 34D);however, heat inactivation of the extracts inhibited their effect,suggesting that the active factor(s) in the extracts was (were)protein(s). Addition of either a polyclonal or monoclonal antibody toHSPB4 suppressed significantly the effect of the necrotic extracts onmacrophage activation, indicating that one of the active factors innecrotic corneal extracts was HSPB4 (FIG. 34D). In addition, recombinantHSPB4 increased the expression of the pro-inflammatory cytokines bymacrophages in a dose-dependent manner (FIG. 34E). As expected, HSPB4caused translocation to the nucleus of the NF-kB complex in macrophages(FIG. 34F). To test whether HSPB4 signaled through the TLR2/NF-kBpathway, a reporter cell line transduced was used to assay TLR2/NF-kBsignaling (HEK-TLR2). Experiments in a HEK-TLR2 cell line demonstratedthat the necrotic extracts of the cornea increased TLR2/NF-kB signalingand that antibodies to HSPB4 inhibited the effect (FIG. 34G).Recombinant HSPB4 stimulated NF-kB signaling in the same cell line in adose-dependent manner (FIG. 34H). HSPB4 acted primarily through TLR2; ithad a smaller effect in the reporter cell line that expressed TLR4 andno effect in the reporter cell without either receptor (FIGS. 341 andJ); however, HSPB4 did not stimulate keratocytes in culture to expresspro-inflammatory cytokines (FIG. 35F). Also, other Group A moleculessuch as HSPB5 and PB-crystallin had no effect on pro-inflammatorycytokine production in macrophages or on NF-kB activation in HEK-TLR2cells (FIG. 34K). In addition, SN did not induce the expression ofpro-inflammatory cytokines in either macrophages or keratocytes in vitro(FIG. 35F). Similarly, HSPB4 did not stimulate keratinocytes in cultureto express pro-inflammatory cytokines. (FIG. 35F). Together, the resultsindicated that HSPB4 was a principal DAMP for Phase II and acted throughthe activation of resident macrophages in the cornea.

TSG-6 Suppressed HSPB4-Induced Activation of Macrophages.

TSG-6 was shown previously to provide a potential therapy for chemicalinjuries of the cornea but its mechanism of action was not established(Oh, Proc. Nat. Acad. Sci., 2010). Therefore, the hypothesis that TSG-6inhibited the Phase II response, but not Phase I (FIGS. 34B and C) wastested by decreasing the HSPB4 induced activation of macrophages.Recombinant TSG-6 decreased expression of pro-inflammatory cytokines inmacrophages stimulated by HSPB4 (FIGS. 35A and B). Because the proteinwas shown previously to inhibit TLR2/NFkB signaling in macrophages byinteraction with CD44 (Choi, Blood, in press; Lesley J, Gal I, Mahoney DJ, et al., TSG-6 modulates the interaction between hyaluronan and cellsurface CD44. J Biol. Chem., 2004; 279:25745-25754). Whether the effectsof TSG-6 were CD44-dependent also were examined As expected, TSG-6 hadno significant effect on NF-kB signaling in the HEK-TLR2 reported cellline unless the cell line were transduced to express CD44 (FIGS. 35C andD); however, TSG-6 had no effect on the parent cell line that did notexpress CD44. Also, TSG-6 had no effect the Phase II inflammatoryresponse after chemical/mechanical injury to the cornea of CD44 knockoutmice (FIG. 35E), indicating that the action of TSG-6 on macrophages wasCD44-dependent.

Example 11 Stanniocalcin-1 Rescued Photoreceptor Degeneration in Two RatModels of Inherited Retinal Degeneration

The purpose of this study was to evaluate the neuroprotective potentialof STC-1 for the therapy of blinding retinal degenerations (RDs) thatinclude photoreceptor degeneration, such as the retinitis pigmentosa(RP) family of inherited RDs and the epidemic atrophic AMD, which is theleading cause of vision loss among the elderly worldwide (Cook et al.,2008). This study demonstrates that intravitreal administration of STC-1rescues photoreceptors from degeneration in two rat models of RD withdifferent etiologies.

Materials and Methods

Animals and Reagents

The experimental protocols were approved by the Institutional AnimalCare and Use Committees of Texas A&M Health Science Center and theUniversity of California, San Francisco.

Recombinant human STC-1 used in this study was purchased from BioVenderResearch and Diagnostic Products (Czech Republic). According to themanufacturer's instructions, distilled water was added to a vial ofSTC-1 that was lyophilized in 20 mM Tris buffer, 20 mM NaCl to yield afinal solution of 0.5 mg/mL.

Vials of frozen passage one hMSCs were obtained from the Center for thePreparation and Distribution of Adult Stem Cells(medicine.tamhsc.edu/irm/msc-distribution.html). Following culture athigh density for 24 hours to recover viable cells, hMSCs were plated atlow density (100 cells/cm²), incubated in complete culture medium (CCM)with 16% FBS for 8 days until approximately 70% confluence was reached,and harvested with 0.25% trypsin/1 mM EDTA at 37° C. for 2 min. Thetrypsin was inactivated by adding the CCM to the cells, and the cellswere washed with PBS by centrifugation at 1,200 rpm for 5 min. The cellswere frozen in α-MEM with 30% FBS and 5% DMSO at a concentration of 1million cells/mL. The same protocol was used to expand the cultures togenerate passage two and then passage three cells. Passage three cellswere used for all experiments. For injection, the cells were lifted,washed by centrifugation with Hank's Balanced Salt Solution (HBSS;BioWhittaker), and suspended in HBSS at a concentration of 20,000cells/μL.

Descriptions of the S334ter-3 and RCS rats can be found at the followingwebsite: www.ucsfeye.net/mlavailRDratmodels.shtml.

Intravitreal Injections

S334ter-3 and RCS rats were anesthetized by isoflurane inhalation.Following topical betadine (5% ophthalmic prep solution, Alcon, FortWorth, Tex.) and proparacaine hydrochloride (0.5% ophthalmic solutionUSP, Bauch & Lomb, Tampa, Fla.), the rats received an intravitrealinjection using a Hamilton syringe (Hamilton #80337) with a 32-gaugeneedle (Hamilton #7803-04). For all experiments in the S334ter-3 rat, 1μg STC-1 (2 μL; 0.5 μg/μL) was injected into the vitreous cavity eitheronce at P9, or twice with one injection at P9 and another at P12.Animals were sacrificed and tissues were collected at P19. Forexperiments with RCS rats, 1 μg STC-1 (2 μL; 0.5 μg/μL) was injected formorphometric and ERG analysis. For real time RT-PCR of transcripts andELISAs for oxidative damage stress, RCS received 2.5 μg STC-1 (5 μL; 0.5μg/μL). Intravitreal injection was performed either once at P21, ortwice at P21 and P28. The rats were sacrificed for tissue collection atP42. For cell therapy experiments, 100,000 hMSCs in 5 μL HBSS wereinjected into the vitreous cavity at P21 and rats were sacrificed fortissue collection at P42. Initiation of treatment for each model wasselected based on the initiation of morphologic degeneration for eachmodel (Martinez-Navarrete et al., 2011; LaVail and Battelle, 1975).Additionally, the volume/dose of STC-1 was based on the age ofinitiation of treatment. For the 5334 rat which was first injected atP9, a maximum of 2 μL was administered. For the RCS which was firstinjected at P21, a maximum of 5 μL was administered due to the largervitreous cavity at this age.

Microarray Assays

A total of 250 ng of RNA from each sample was applied for microarraysusing GeneChip 3′IVT Express Kit (Affymetrix) according tomanufacturer's directions. Briefly, poly-A RNA controls were added intoeach sample to provide exogenous positive controls to monitor theeukaryotic target labeling process. T7 oligo(dT) primer was used togenerate first strand cDNA followed by second strand cDNA synthesis. Togenerate biotin modified aRNA, in vitro transcription was performedfollowed by purification and quantification of labeled aRNA. A total of15 μg of aRNA was fragmented and hybridized (GeneChip Hybridization Oven640; Affymetrix) onto rat arrays (RG230 2.0, Affymetrix) followed byarray washing and staining (GeneChip Fluidics Station 450; Affymetrix)with GeneChip Hybridization, Wash, and Stain Kit (Affymetrix). Arrayswere scanned with GeneChip Scanner (Affymetrix) and images checked forquality. Data were normalized using robust multi-array (RMA) algorithmand gene level analysis was performed with Partek Genomics Suite 6.4(Partek). To obtain up- and down-regulated genes, STC1-treated sampleswere compared with PBS-treated samples within each model and expressionlevel changes of at least 1.5-fold were considered significant. Genesupregulated at least 1.5-fold in both models were used for detection ofenriched Gene Ontology terms using the Partek Genomics Suite 6.4software.

Real Time RT-PCR Assays

For RNA extraction, retinas were isolated by surgical excision,immediately placed in RNA isolation reagent (RNA Bee, Tel-Test Inc.,Friendswood Tex.), and frozen at −80° C. The samples were rapidly thawedand homogenized on ice, and total RNA was extracted (RNeasy Mini kit;Qiagen). cDNA was generated by reverse transcription (SuperScript III;Invitrogen) using 1 μg total RNA. Real time amplification was performedusing TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad,Calif.). PCR probe sets and Taqman Gene Expression Assay kits (AppliedBiosystems, Carlsbad, Calif.) were used to measure gene expression (Rho:Rn00583728 m1; Rcvrn: Rn00590194 m1; Pdc: Rn00563505 m1; NRLZ:Rn01502072g1; UCP-2: Rn01754856_m1; and 18s: 4352930E). Values werenormalized to 18s RNA and expressed as a fold change compared to thefellow eye.

Histological Analysis

Tissue processing and histological analysis was performed as previouslydescribed (Lewin et al., 1998). Briefly, following euthanasia byoverdose of carbon dioxide, eyes were enucleated and immediately fixedin a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde. Eyes wereembedded in epoxy resin, and 1 μm-thick sections were made along thevertical meridian. To quantify photoreceptor loss, a mean ONL thicknesswas obtained by taking an average of a total of 54 measurements from thesuperior and inferior hemispheres (27 per hemisphere) using the BioquantMorphometry System (R & M Biometrics Inc., Nashville, Tenn.). Pyknoticindex was determined by counting numbers of pyknotic photoreceptornuclei as a percent of total photoreceptor nuclei.

Electroretinogram Analysis

Electroretinography was performed as previously described (Lewin et al.,1998). Briefly, following overnight dark-adaption, rats wereanesthetized with ketamine (80 mg/kg) and xylazine (16 mg/kg). Pupilswere dilated with 1% tropicamide and 2.5% phenylephrine. ERGs wererecorded using a wire contacting the corneal surface with 1%methylcellulose. The signal was amplified, digitized, and stored usingan LKC UTAS-3000 Diagnostic System (Gaithersburg, Md.).

ELISAs for Markers of Oxidative Damage

For protein extraction, retina was sonicated on ice (UltrasonicProcessor, Cole Parmer Instruments, Vernon Hills Ill.) in a Tris-EDTAsolution containing protease inhibitor cocktail (Roche, Indianapolis,Ind.). After centrifugation at 12,000 rpm at 4° C. for 20 min, thesupernatant was assayed for protein carbonyl content (OxiSelect™ ProteinCarbonyl ELISA Kit, Cell Biolabs, Inc., San Diego Calif.) ornitrotyrosine content (OxiSelect™ Nitrotyrosine ELISA Kit, Cell Biolabs,Inc. San Diego, Calif.).

Real Time RT-PCR Based Standard Curve for hGAPDH

A standard curve was generated by adding serial dilutions of hMSCs torat tissue as previously described (Lee et al., 2009). Briefly, 100 to100,000 hMSCs were added to each rat whole globe. Following RNAextraction (RNeasy Mini kit; Qiagen, Valencia, Calif.), cDNA wasgenerated by reverse transcription (SuperScript III; Invitrogen,Carlsbad, Calif.) using 1 ng total RNA. Human-specific GAPDH primers andprobe (TaqMan Gene Expression Assays ID, GAPDH HS99999905_(—)05) wereused. The standard curve was made based on hGAPDH expression from aknown number of hMSCs added to one rat globe and the values werenormalized to total eukaryotic 18s rRNA (TaqMan Gene Expression Assays,4352930E).

Statistics

Paired, 2-tailed Student's t-tests were used to compare treated andcontrol eyes from the same rat in all experiments.

Results

STC-1 in the S334Ter-3 Rat: Improved Survival of Photoreceptors

To test the therapeutic effects of STC-1, a model in which there israpid degeneration of both rod and cone photoreceptors, the S334ter-3rhodopsin transgenic rat (Liu et al., 1999; Martinez-Navarrete, 2011)was first selected. The early onset of photoreceptor degeneration isseen as an increase in the incidence of pyknotic photoreceptor nuclei inthe outer nuclear layer (ONL) beginning at postnatal day 8 (P8), withmany more pyknotic nuclei present and obvious disorganization ofphotoreceptor inner segments at P10, just as photoreceptor outer segmentdevelopment begins (Liu et al., 1999). Photoreceptor cell loss is thenrapid, and only a single row of photoreceptor nuclei remains in thecentral retina at P20 (Liu et al., 1999), and no rod outer segments everdevelop in the S334ter-3 rat (Liu et al., 1999; Martinez-Navarrete etal., 2011). It was elected to test a maximal dose of STC-1 based on theconcentration of the undiluted commercially supplied protein (0.5 μg/μl)and volume tolerated for injection into the vitreous. Therefore, 1 μgSTC-1 was injected into the vitreous cavity either once at P9, or twicewith one injection at P9 and another at P12. In eyes treated with STC-1(FIGS. 37A, C, and D; Table 15), there was an increased number ofsurviving photoreceptor nuclei on P19 as assayed by quantitative measureof the ONL thickness (Lewin et al., 1998).

TABLE 15 Intravitreal administration of STC-1 rescued photoreceptors inthe S334ter-3 rat. Average measurements of the thickness of outernuclear layer (ONL) in μm in mean superior (Mean Sup) and mean inferior(Mean Inf) hemispheres from 15 eyes receiving STC-1 (X1) and 10receiving STC-1 (X2) and their corresponding contralateral controlsshowed significant rescue following one or two injections of STC-1.Error bars represent means ± standard deviation. ONL thickness (μm) No.of Mean Mean Mean animals Sup Inf total Uninjected 15 6.9 ± 1.1 8.8 ±1.0 7.9 ± 1.0 STC-1 injected 15 11.2 ± 2.3  12.5 ± 2.3  11.8 ± 2.2  (X1)P-Value 0.0000001 0.00003 0.000001 Uninjected 10 6.7 ± 0.5 7.8 ± 1.3 7.3± 0.7 STC-1 injected (X2) 10 11.4 ± 2.7  12.0 ± 2.5  11.7 ± 2.4  P-Value0.0005   0.0002  0.0002 

Similar results were obtained with one injection (n=15, P=0.000001) ortwo injections (n=10, P=0.0001) (Table 15). In the histologicalanalysis, no apparent negative effect of STC-1 was found.

STC-1 in S334Ter-3 Rat: Improved Retinal Function

To test functional improvements in the retina following STC-1 therapy,eyes of the rats were examined with electroretinography (ERG), whichprovides a measure of rod- and cone-generated responses to varyingstimuli of light. The responses from dark-adapted retinas (scotopic) areprimarily from rods, and those from light-adapted retinas (photopic) areprimarily from cones. ERG responses to light stimuli greater than noiselevels require the transduction of light by photoreceptor outersegments. As noted above, rod outer segments never develop in theS334ter-3 rats (Liu et al., 1999; Martinez-Navarrete et al., 2011), andusing PNA staining, only a very few, short cone outer segments are foundin the central retina of S334ter-2 rats (Martinez-Navarrete et al.,2011). As a consequence, ERG response amplitudes are almost never seenabove threshold levels of 20 μV for the scotopic b-wave and 10 μV forthe photopic b-wave in this line of rats. However, following a singleinjection of STC-1 at P9, the amplitudes of both the scotopic andphotopic ERG responses at P19 were greater than threshold levels andgreater than those in control eyes in the same animals (FIG. 37B).Therefore, STC-1 treatment results in a functional improvement of bothrod and cone photoreceptors.

STC-1 in the S334Ter-3 Rat: Improved Levels of Photoreceptor mRNAs

For quantitative assays of photoreceptor viability, surveys withmicroarrays to identify candidate genes whose expression decreased withtime in the retinas of the rats were first selected (Table 16 and Table17).

TABLE 16 Selected Gene Ontology terms enriched in the group ofup-regulated genes in S334ter-3 and RCS rat retinas followingintravitreal administration of STC-1. Genes up- regulated at least1.5-fold by STC-1 treatment in both models were used for detection ofenriched Gene Ontology terms (Partek Genomics Suite 6.4 software). No.of Selected Gene Families (Listed by enrichment p-value) genes in list P< 0.0001 phototransduction 9 detection of light stimulus involved invisual perception 9 photoreceptor cell development 6 rhodopsin mediatedphototransduction 3 neuron development 6 photoreceptor outer segment 4nonmotile primary cilium 4 transcription 5 photoreceptor cellmorphogenesis 2 P < 0.001 neurological system process 8 cell development7 intracellular cyclic nucleotide activated cation 2 channel activity P< 0.01 photoreceptor cell maintenance 2 negative regulation of caspaseactivity 2 ligand-gated ion channel activity 3 transmembrane transporteractivity 5 response to stimulus 17  P < 0.05 anatomical structurehomeostasis 3 presynaptic membrane 2 cell morphogenesis involved indifferentiation 2 gated channel activity 3 positive regulation oftranscription from 5 RNA polymerase II promoter channel activity 3passive transmembrane transporter activity 3 cellular hormone metabolicprocess 2 cellular developmental process 9

TABLE 17 Selected genes of interest up-regulated in S334ter-3 and/or RCSrat retinas following intravitreal administration of STC-1. Selectedgenes up-regulated over 2-fold compared to contralateral controls. Foldchange S334 Reference Gene Name ter-3 RCS Function (PMID) Aipl1 Arylhydrocarbon receptor — 2.0 Required for assembly of functional 19758987interacting protein-like 1 rod phosphodiesterase subunits Cabp4 Calciumbinding protein 4 2.2 2.8 Retinal cone and bipolar cell 16249514development Cngb1 Cyclic nucleotide gated 2.0 2.4 Subunit of the cyclicnucleotide- 7682292 channel beta 1 gated cation channel in rods CrxCone-rod homeobox — 2.6 Coordinates photoreceptor gene 20693478expression Drd4 Dopamine receptor 4 — 2.0 Suppresses adenylate cyclasein the 12763097 retina Fabp12 Fatty acid-binding protein 2.1 3.3 Subunitof rod channel 18786628 12 Gnat1 Guanine nucleotide 2.2 3.9 Subunit of Gprotein which stimulates 17584859 binding protein, alpha the coupling ofrhodopsin and cGMP- transducing activity phoshodiesterase during visualpolypeptide 1 impulses. Gnb3 Guanine nucleotide- — 2.2 Isoform of thebeta subunit of the 20538044 binding protein beta3 heterotrimeric Gprotein second messenger complex. Expressed in photoreceptors GrkS Gprotein-coupled 2.1 — Phosphorylates rhodopsin 8120045 receptor kinase 5Guca1a Guanylate cyclase — 2.0 Ca2+ dependent negative feedback 19459154activating protein 1 regulation of membrane bound guanylate cyclases inrods and cones Kcnv2 Potassium channel, 2.2 — Voltage-gated potassiumchannel in 16909397 subfamily V, member 2 rods and cones Lgals3 Lectin,galactoside- 2.2 — Expressed in Muller cells 19816601 binding, soluble,3 Lrp5 Low-density lipoprotein 2.1 — Muller cell function 20652025receptor-related protein 5 Nrl Neural retina leucine 2.0 3.1 Regulatorof photoreceptor 1729696 zipper differentiation and function Nxnl1Nucleoredoxin-like 1 — 2.3 Defense against oxidative stress 21079812Involved in cone survival 20949100 Pdc phosducin — 2.2 Regulatestransmission at the 20203183 photoreceptor-to-ON-bipolar cell synapsePde6b Phosphodiesterase 6B, — 2.4 Composes catalytic subunit of key20940301 cGMP-specific, rod, beta effector enzyme of thephototransduction cascade Prph2 Peripherin 2 — 2.7 Necessary for conestructure 1071739 Rcvrn Recoverin 2.5 3.0 Termination of thephototransduction 1672047 cascade in the retina Reep6 Recoverin 2.1 3.0Termination of the phototransduction 1672047 cascade in the retina RhoRhodopsin — 3.2 G-protein-coupled receptor 20708633

Real time RT-PCR assays were used for transcripts expressed inphotoreceptors. Beginning on about P12, there was a rapid two-daydecrease in the levels of mRNAs for four photoreceptor specific genes:rhodopsin, recoverin, phosducin, and neural retina leucine zipper (FIG.38A). The decrease in the mRNAs occurred with a similar time course aspreviously observed by morphology of photoreceptor loss in thetransgenic rats (Liu et al., 1999). The slight increase in photoreceptorgene expression observed between P8 to P12 is consistent with theobservations that photoreceptor development continues to increasepostnatally until about P12 (Chiang and Barnstable, 1998). A singleintravitreal injection of STC-1 significantly increased the levels ofmRNAs for three of the genes: rhodopsin, phosducin, neural retinaleucine zipper (FIG. 38B). Two injections significantly increased thelevels of all four photoreceptor genes compared to uninjected (UI)controls. Additionally, there appeared to be a dose-dependent trend inrescue of photoreceptor transcripts.

STC-1 in the RCS Rat: Improved Survival of Photoreceptors

Next the effects of STC-1 were examined in a slower model of RD, theRoyal College of Surgeons (RCS) rat (LaVail and Battelle, 1975) which ischaracterized by a dysfunctional RPE that is unable to remove shed outersegments of photoreceptors that accumulate and lead to the death of thephotoreceptors. Additionally, other debris including pyknoticphotoreceptor nuclei remain due to impaired phagocytic clearing (LaVailand Battelle, 1975). One ng STC-1 was injected into the vitreous cavityeither once at P21, or twice with one injection at P21 and another atP28. Quantitative morphometric analysis of the thickness of the ONL atP42 did not show any difference between control eyes and STC-1 injectedeyes. However, STC-1 produced a marked improvement in morphology asreflected by longer photoreceptor inner segments and fewer pyknoticnuclei in the STC-1 treated eyes compared to UI controls (FIGS. 39A andB; Table 2).

TABLE 18 Intravitreal administration of STC-1 rescued photoreceptors inthe RCS rat. Pyknotic index showed a decreased percent of pyknoticnuclei in STC-1 treated eyes after single injection on P22 and assay onP42, injections on P21 and P28 with assay on P42, or injection on P25and assay on P51. Pyknotic index was determined as the proportion of thenumber of pyknotic photoreceptor nuclei to the number of all nuclei inthe ONL in ten 440- μm length sections of posterior retina from eachanimal (5 in the inferior hemisphere and 5 in the superior hemisphere).Error bars represent means ± standard deviation. Age(s) Injected- No. ofSTC-1 Eyes taken animals Uninjected (%) injected (%) P-Value P22-P42 975.0 ± 3.5 47.8 ± 12.3 <0.0005 P21, P28-P42 6 67.5 ± 4.2 26.7 ± 16.0<0.005 P25-P51 7 60.0 ± 2.9 34.6 ± 19.5 <0.05In the UI control eyes, many of the pyknotic nuclei coalesced with othernuclei to form large masses of chromatin, an observation previouslydescribed in the RCS rat (LaVail and Battelle, 1975) and the similar Merknockout mouse (Duncan et al., 2003). The delay in clearance of pyknoticphotoreceptor nuclei in the RCS rat is due to a null mutation in thegene for c-mer proto-oncogene tyrosine kinase (Mertk) that is expressednot only in RPE cells that phagocytose shed outer segments ofphotoreceptors, but also in macrophages such as those that invade theRCS retina (LaVail, 1979) and that show a generalized defect in removalof some apoptotic cells (Scott et al., 2001). Of note, a singleinjection of STC-1 produced these morphologic improvements 26 days aftertreatment providing evidence that the effects are relativelylong-lasting. As with the S334ter-3 rats, no negative effects caused bySTC-1 in the RCS rats was found.STC-1 in the RCS Rat: Improved Retinal Function

To test functional improvements following STC-1 therapy, ERG responseamplitudes were measured at P42 in RCS rats that received one injectionat P21 and a second injection at P28. In the RCS rat, significantimprovements in scotopic ERG responses were observed (FIG. 39C).Photopic responses were not improved in the RCS rat; however, this isnot surprising as cone degeneration does not begin in the RCS rat untilapproximately P44 (Pinilla et al., 2004).

STC-1 in the RCS Rat: Improved Levels of Photoreceptor mRNAs

As expected, real time RT-PCR assays demonstrated a more gradual declinein photoreceptor gene expression in the RCS rat (FIG. 40A) than in theS334ter-3 rat (FIG. 38A). The rate of loss was similar to the rate ofmorphological changes previously observed in the retina (LaVail andBattelle, 1975). Intravitreal injection of STC-1 increased the levels ofthree of the photoreceptor transcripts in the eye at P42 after a singleintravitreal injection of 2.5 ng of STC-1 at P21 and of all fourphotoreceptor transcripts after two injections, one injection at P21 anda second at P28 (FIG. 40B), compared to PBS-injected controls.Additionally, there appeared to be a dose-dependent trend in the rescueof photoreceptor transcripts.

STC-1 in the RCS Rat: Decrease in Products of ROS

To test the hypothesis that the improvements of photoreceptor viabilitywere explained by the ability of STC-1 to upregulate UCP-2, expressionof UCP-2 was assayed by real time RT-PCR following intravitrealinjection of STC-1 in the RCS rat. Significant increases in UCP-2 geneexpression were observed in the retina three days following injection ofSTC-1 at P21 (FIG. 41A). For evidence of a reduction in oxidative stressfollowing STC-1 treatment, the retinas were assayed for protein carbonyland nitrotyrosine, products of ROS that were used previously to measureoxidative damage in models of RD (Usui et al., 2009; Komeima et al.,2008) and were observed in the eyes of patients with AMD (Murdaugh etal., 2010; Ng et al., 2008). Assays of retinas at P42 demonstrated thatinjection of STC-1 at P21 and P28 decreased the levels of both proteincarbonyl and nitrotyrosine at P42 (FIGS. 41B and C) compared toPBS-injected controls.

STC-1 in Both Models: Common Gene Expression Changes

To make global comparisons between the two models, RNA from S334ter-3and RCS retinas were analyzed by gene expression microarrays. Visualcomparisons of heat maps demonstrated major differences between the twomodels. However, the data also indicated that administration of STC-1produced up-regulation of a series of identical genes in both models(Table 16, Table 17). Both models showed significant up-regulation ofimportant recovery associated genes with functions in phototransduction,photoreceptor cell development and morphogenesis, neuron development,negative regulation of caspase activity, and in other related processes(Table 16, Table 17). Additionally, a smaller class of genes wasdown-regulated following STC-1 injection in both models (Table 19).

TABLE 19 Genes down-regulated in both S334ter-3 and RCS rat retinasfollowing intravitreal administration of STC-1. Fold change Gene NameS334ter-3 RCS Gjb2 Gap junction protein, beta −2.2 −2.1 2 Agr2 Anteriorgradient homolog −2.5 −2.2 2 Dcn Decorin −6.6 −3.6Human MSCs as a Vehicle to Deliver STC-1 in the RCS Rat

The hypothesis that intravitreal administration of human MSCs (hMSCs)could be used to deliver STC-1 to the retina was also tested. Previousreports demonstrated that MSCs rescued photoreceptor degeneration inmodels of RD following subretinal injection (Inoue et al., 2007; Kicicet al., 2003) or intravenous infusions of the cells (Wang et al., 2010).Recent data from our laboratory suggested that in disease modelscharacterized by apoptosis and oxidative stress, MSCs exert theirtherapeutic effects at least in part by being activated by signals frominjured tissues to increase expression of the anti-apoptotic/anti-ROSprotein STC-1 (Block et al., 2009).

Injection of the hMSCs into the eyes of RCS rats at P21 significantlyincreased levels of transcripts for rhodopsin, phosducin, recoverin, andneural retinal leucine zipper at P42 (FIG. 43). The effects observedwere similar to those observed following treatment with STC-1.Therefore, the hypothesis that the effects observed could be explainedby the cells remaining viable in the rat vitreous cavity to increaseexpression of STC-1 was tested.

To follow the fate of the hMSCs following intravitreal injection, ahuman-specific real time RT-PCR assay for mRNA for human GAPDH (hGAPDH)was used (FIG. 42A) (Lee et al., 2009). After intravitreal injection of100,000 hMSCs into the eyes of RCS rats, the assay for hGAPDH mRNAindicated that 10% of the injected cells were recovered after 5 days and1% of the injected cells after 21 days (FIG. 42B). Real time RT-PCRassays also indicated that at day 5, the surviving hMSCs expressed humanSTC-1. The level of expression of STC-1 per human MSC recovered from thevitreous was about 10-fold higher than in the preparation of hMSCs thatwas not injected. (FIG. 42C). Therefore the results were anotherillustration of how hMSCs are activated to express therapeutic genes inresponse to cross-talk with injured cells and tissues (Lewin et al.,1998). The results may in part provide a molecular explanation for theresults observed by others that MSCs reduced degeneration in the retinafollowing subretinal (Inoue et al., 2007; Kicic et al., 2003) orintravenous (Wang et al., 2010) administration. The results alsoprovided evidence that hMSCs could be used to deliver STC-1 in settingswhere repeated therapeutic injections are not indicated. Based onquantitative gene expression data, a single injection of hMSCs appearedto have a rescue effect superior to a single injection of STC-1, butless than the effects observed with two injections of STC-1. Therefore,it was decided to pursue the more clinically conservative therapy ofprotein injection since repeated intravitreal protein injections (e.g.,anti-VEGF therapy) are now commonly used in the clinical setting forocular neovascular disease (Tolentino, 2011).

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

REFERENCES

-   Aggarwal et al., 2005, Blood 105:1815-22-   Akiyama et al., 2002, Glia 39(3): 229-36-   Armstrong et al., 1998, Angiogenesis. 2(1):93-104.-   Avila et al., 2001, Cornea 20:414-20-   Block et al., 2009, Stem Cells 27(3):670-81-   Callaghan et al., 2001, Rheumatology (Oxford) 46:105-11-   Caplan, 1990, Biomaterials 11:44-6-   Caplan et al., 2005, Tissue Eng 11:1198-1211-   Caplan et al., 2006, J Cell Biochem 98(5):1076-84-   Castro-Malaspina et al., 1980, Blood 1980 56(2):289-301-   Cauchi et al., 2008, Am J Ophthalmol 146:251-9-   Cho et al., 1998, Cornea 17:68-73-   Chen et al., 2006, Immunol Cell Biol 84:413-21-   Clegg et al., 2006, Ophthalmic Epidemiol 13:263-74-   D'Amato et al., 1994, Proc Natl Acad Sci USA 91:4082-5-   Daya et al., 2005, Ophthalmology 112:470-7-   De Ban et al., 2003. J Cell Biol 160(6) 909-18-   Dooner et al., 2004, Blood Cells Mal Dis 32(1):47-51-   Eaves et al., 2001, Ann N Y Acad Sci 938:63-70, discussion 70-1-   Espana et al., 2003, Br J Ophthalmol 87:1509-14-   Fantes et al., 1990, Arch Ophthalmol 108:665-75-   Foster C S, Letko E, Ba-Abbad R A Stevens-Johnson Syndrome    [Internet] [updated 2007 Dec. 18, cited 2008 March 7] Available from    http://emedicine.medscape.com/article/1197450-overview-   Fukuda et al., 2006, Circ Res 98(8): 1002-13-   Gao et al., 2001, Cells Tissues Organs 169(1):12-20-   Gerdoni et al., 2007, Ann Neurol 61(3):219-27-   Guilak et al., 2004, Biorheology 41(3-4):389-99-   Gupta et al., 2007, J Immunol 179(3):1855-63-   Hogg et al., 1994, J Appl Physiol 77(4): 1795-800-   Homma et al., 2004, Invest Ophthalmol Vis Sci 45:4320-6-   Horwitz et al., 2002, Proc Natl Acad Sci USA 99(13):8932-7-   Horwitz et al., 1999, Nat Med 5:309-13-   Ilari et al., 2002, Ophthalmology 109:1278-84-   Iso et al., 2007, Biochem Biophys Res Commun 354:700-6-   Javazon et al., 2001, Stem Cells 19:219-25-   Jenkins et al., 1993, Eye 7:629-33-   Kim et al., 2006, Brain Res 1123(1):27-33-   Koc et al., 2002, Bone Marrow Transplant 30:215-22-   Krampera et al., 2007, Bone 40(2):382-90-   Kuznetsov et al., 2001, J Cell Biol 153(5):1133-40-   Le Blanc et al., 2007, J Intern Med 262(5):509-25-   Lee et al., 2006, Proc Natl Acad Sci USA 103:17438-43-   Lee et al., 2009, Cell Stem Cell 5:54-63-   Limb et al., 2008, Br Med Bull 85:47-61-   MacDonald et al., 2002, Bioessays 24(10):885-93-   Mareschi et al., 2006, Exp Hematol 34(11) 1563-72-   Melsaether C, Rosen CL Burns. Ocular [Internet] [updated 2009 August    12] Available from    http://emedicine.medscape.com/article/798696-overview-   Mertzants et al., 2005, Invest Ophthalmol V is Sci 46:46-50-   Mets et al., 1981, Mech Ageing Dev 16(1):81-9-   Mitjanovic et al., 2007, Am J Ophthalmol 143:409-15-   Milner et al., 2003, J Cell Sci 116:1863-73-   Milner et al., 2006, Biochem Soc Trans 34:446-50-   Mishra, 2008, J Cardiovasc Med (Hagerstown) 9(2):122-8-   Moss et al, 2000, Arch Ophthatmol 118:1264-8-   Moss et al., 2008, Optom V is Sci 85:668-74-   Munoz et al., 2005, Proc Natl Acad Sci USA 102:18171-6-   Nomura et al., 2005, Neuroscience 136(1):16t-9-   Oh et al., 2008, Stem Cells 26:1047-55-   Oh et al., 2009b, Curr Eye Res 34(2):85-91-   Oh et al., 2009a, Cytokine 46(1)100-3-   Ohtaki et al., 2008, Proc Natl Acad Sci USA 105:14638-43-   Ortiz et al., 2007, Proc Natl Acad Sci USA 104:11002-7-   Owen et al., Ciba Found Symp 136:42-60-   Penolazzi et al., 2008, Cell Biol Int 32:320-5-   Pereira et al., 1998, Proc Natl Acad Sci USA 95(3):! 142-7-   Pflugfetder et al., 2008, Am J Manag Care 14:S102-S106-   Piersma et al., 1983, Br J Haematol 54(2):285-90-   Prockop, 1985, J Clin Invest 75(3):783-7-   Prockop et al., 1995, Annu Rev Biochem 64:403-34-   Prockop, 1997, Science 276:71-4-   Prockop et al., 2003, Proc Natl Acad Sci USA 100; 119 17-23-   Prockop, 2007, Clin Pharmacol Ther 82:241-3-   Prockop, 2009, Mol Ther 17:939-46-   Rao et al., 1999, Ophthalmology 106:822-8-   Reddy et al., 2004, Cornea 23:751-61-   Ren et al., 2008, Cell Stem Cell 0.2:141-50-   Reinhard et al., 2004, Ophthalmology 111:775-82-   Ringden et al., 2006, Transplantation 81:1390-7-   Rosada et al., 2003, Calcif Tissue 72(2):135-42-   Schaumberg et al., 2003, Am J Ophthalmol 136:318-26-   Schinkothe et al, 2008, Stem Cells Dev 17:199-206-   Schrepfer et al., 2007, Transplant Proc 39(2) 573-6-   Seo et al., 2005, J Dent Res 2005 84(10):907-12-   Sharpe et al., 2007, Tissue Eng 13:123-32-   Shi et al., 2008, Clin Exp Ophthalmol 36:501-7-   Shorn et al., 2007. Sury Ophthalmol 52:483-502-   Solomon et al., 2002, Ophthalmology 109:1159-66-   Spees et al, 2006, Proc Natl Acad Sci USA 103(5):1283-8-   Tang et al., 2007, Cell Transplant 16(2)159-69-   Ti et al., 2002, Invest Ophthalmol V is Sci 43:2584-92-   Tseng et al., 1998, Arch Ophthalmol 116:431-41-   Tsubota et al., 1999 N Engl J Med 340:1697-703-   Ueno et al., 2007, Cornea 26:1220-7-   Wakitani et al., 1995, Muscle Nerve 18(12): 1417-26-   Wisniewski et al, 2004, Cytokine Growth Factor Rev 15: 129-46-   Woodbury et al., 2000, J Neurosci Res 61(4):364-70-   Wu et al, 2008, Cell Transplant 16(10):993-1005-   Zacharek et al., 2007, J Cereb Blood Flow Metab 27:1684-91

What is claimed is:
 1. A method for reducing corneal inflammation, orpromoting epithelial wound healing in the eye, or reducing photoreceptordegeneration, or a combination thereof in a subject in need thereof,comprising administering to the subject a pharmaceutically effectiveamount of a composition that comprises an isolated polypeptidecomprising a domain comprising a stanniocalcin family memberpolypeptide, wherein the stanniocalcin family member polypeptide is aSTC-1 polypeptide that has at least 95% sequence identity to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. 2.The method of claim 1, wherein the stanniocalcin family memberpolypeptide has at least 95% sequence identity to SEQ ID NO:1.
 3. Themethod of claim 2, wherein the stanniocalcin family member polypeptidecomprises SEQ ID NO:1.
 4. The method of claim 1, wherein the compositionis administered intravitreally, subconjuntivally or topically to theeye.